Replace Toxic Products in your home

Everyday Roots

This book includes home remedies, natural beauty recipes and Diy household product tutorials. Discover over 215 suprising natural home remedies using common ingredients like onion, lemons and apple cider vinegar. EveryDay Roots will help you to make healthy changes in your life. Learn how to treat coughs, headaches and other health conditions with common ingredients like honey and watermelon. When you buy the book you get a 328 page Pdf with a clickable table of contents. Continue reading...

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Protein Elution From Pvdf Membranes Using Detergents

Binding affinities of most proteins to PVDF membranes are relatively strong. The most efficient protocol for protein recovery from PVDF membranes requires the use of detergents, which limits the possible use of extracted samples because detergents are often incompatible with subsequent procedures. This protocol is a simple procedure to elute proteins from the membrane into a Triton SDS solution. A protocol that employs acidic extraction with organic solvents is also described (Alternate Protocol 4).

Analysis of Membrane Proteins in Detergents

The separation of membrane proteins in the presence of detergents (typically a nonionic or zwitterionic detergent) is the same in principle as separation in aqueous solutions. However, in the presence of detergent, the apparent size of the protein will probably increase due to detergent binding to the protein. For this reason, the fractionation range of gel filtration columns specified for water-soluble proteins will not apply to detergent-solubilized proteins (Schagger, 1994). Accurate size determination is not feasible in the presence of detergents, since membrane proteins will bind detergent molecules and or interact with detergent micelles to differing degrees. The procedure for gel filtration in the presence of detergent is essentially as described in the Basic Protocol, with a few additional factors to take into account. First, the protein sample should be equilibrated with the detergent prior to the run. Detergents with a low critical micelle concentration (CMC) do not dialyze...

Detection Of Tissue Transglutaminase Activation By Cell Resistance To Detergents

This protocol is a simple and rapid approach to identify apoptotic cells with activated TGase 2. The method is based on the propensity of crosslinked protein to withstand treatment with detergents. The authors have noticed that when live, nonapoptotic cells are subjected to treatment with solutions of nonionic detergents, lysis of their plasma membrane and release of the content of cytoplasm is complete, resulting in preparation of isolated nuclei. In contrast, apoptotic cells resist the detergent treatment their cyto-plasmic protein remains insoluble, attached to the nucleus in the form of a shell-like cover (Grabarek et al., 2002). It is possible, therefore, by flow or laser scanning cytometry to distinguish apoptotic cells from the nuclei isolated from nonapoptotic cells, by means of the abundance of protein in the former. In addition, bivariate gating analysis of cellular DNA and protein content makes it possible to reveal the cell cycle distribution separately for the population...

Choice Of Detergents

Ionic detergents are very good solubilizing agents, but they tend to denature proteins by destroying native three-dimensional structures. This denaturing ability is useful for SDS-PAGE (unit 8.4), but is detrimental where native structure is important, as when functional activities must be retained (antibody activity is usually retained in < 0.1 SDS). Nonionic and mildly ionic detergents are less denaturing and can often be used to solubilize membrane proteins while retaining protein-protein interactions. The following detergent properties are detrimental in certain procedures 1. Phenol-containing detergents (e.g. Triton X-100 and NP-40) have a high absorbance at 280 nm and hence interfere with protein monitoring during chromatography (most ionic detergents do not absorb at 280 nm Brij- and Lubrol-series detergents are nonionic detergents that do not have substantial absorbance at 280 nm). Phenol-containing detergents also induce precipitation in the Folin protein assay (but they...

Types Of Detergents

A large variety of detergents are available (Helenius et al., 1979). For biochemical studies, they are usually categorized according to the type of hydrophilic group they contain anionic, cationic, amphoteric, or nonionic. Tables A.1B.1 and A.1B.2 list commonly used members of each type. In general, nonionic and amphoteric detergents are less denaturing for proteins than ionic detergents. Sodium cholate and sodium deoxycholate are the least denaturing of the commonly used ionic detergents. Two properties of detergents are important in their consideration for biological studies the critical micelle concentration (CMC) and the micelle molecular weight (Table A.1B.1). The CMC is the concentration at which monomers of detergent molecules combine to form micelles each detergent micelle has a characteristic micelle molecular weight. Detergents with a high micelle molecular weight, such as nonionic detergents, are difficult to remove from samples by dialysis. The CMC and the micelle...

Membraneassociated And Insoluble Nonrecombinant Proteins

Proteins that are not physiologically soluble can be purified after extracting and removing soluble proteins, thereby achieving a substantial degree of purification at the extraction step (Fig. 1.3.4 also see units6.1 & 6.2). To carry out a purification it is nearly always necessary to obtain the desired protein in a soluble form, which will often require the addition of solubi-lizing agents such as detergents. Some proteins remain insoluble even with detergent treatment, and so can be substantially purified by removing the soluble fractions. Some membrane-associated proteins become partly solubilized during breaking up of the tissue, and recovery in the particulate fraction may be poor. In such cases it may be best to solubilize the whole tissue by including detergent in the homogenizing buffer. Extraction of insoluble residues using detergents can be done differentially some proteins are released at low detergent concentration, whereas others require complete solubilization of...

Limitations Of The In Vitro Manipulation Of Proteins

The experimental manipulation of membrane proteins is decidedly more difficult. The exterior face exists in a high Na+ and Ca2+ solution that is oxidative just the opposite is true for the cytoplasmic face. Since no appropriate solvent exists for such isolated proteins, experimental questions can be directed at properties associated with just one face of the molecule. A second limitation common to all in vitro studies of transmembrane proteins is that they must be solubilized using detergents. In addition to solubilizing a transmembrane protein, detergents can also bind to hydrophobic regions in the globular domain(s) of the protein, thus affecting the properties under study. One means of countering this problem is to test several detergents in the hope of finding one that retains the full biological activity of the purified protein. valently cross-link protein assemblies prior to disruption and to solubilize proteins using mild detergents (e.g., Brij-58).

Incorporation Of Integral Membrane Proteins Into Iscoms

Preparation of membrane fractions is outside the scope of this manual (see Helenius and Simons, 1975). However, it is important to ensure that the protein(s) of interest is soluble in the detergent used to extract the membrane material and that the detergent is dialyzable. Octyl glucoside, Mega 10, or related detergents are most suitable (see Table A.1D.1). Precautions should also be taken to prevent proteolytic degradation of the protein during extraction.

Mass Spectrometry Identification of Proteins 302221 Overview of the instrumentation

Depending on the instrumental setup, protein identification by MS employs mass or sequence information or a combination of the two. A classical application of MALDI-ToF instruments is the so-called peptide mass fingerprinting (PMF). In PMF the masses of the tryptic peptides derived from a protein are measured, and the list of peptides masses is submitted to a database, in which proteins are stored as a collection of peptides according to the cleavage specificity of trypsin. It is the combination of a large enough number of accurately measured peptide masses that allows unambiguous identification of proteins based only on this 'fingerprint,' without having any sequence information. The importance of mass accuracy was nicely illustrated by Clauser et al.45 using a peptide mass fingerprint set consisting of 23 peptide masses derived from a 2D gel spot of bovine apolipoprotein A I (Apo A I). When interrogating the database (GenBank translated into proteins), assuming a mass accuracy of 2...

The Protein Based Approach Two DimensionalPolyacrylamide Gel Electrophoresis

One inherent advantage of 2D-PAGE is that it is performed under denaturing conditions and hence the risk of proteomic changes during sample preparation, e.g., by remaining enzymatic activity in the sample, is very limited. A typical sample buffer for 2D-PAGE contains chaotropes, such as (thio)urea and nonionic or zwitterionic detergents like (CHAPS) to improve the solubilization of proteins and the transfer of proteins to the second-dimension gel.50

Selective Precipitation Using Hydrophobic Ion Pairing Hip Entanglement Ligands

Hydrophobic ion pairing (HIP) is coprecipitation of proteins using flexible hydrocarbon tails of alkane anions (detergents) that have their anion head groups bound in the target proteins. Figure 4.5.8 illustrates the composition of such a coprecipitate. Organic anions, especially sulfonates and sulfates, often bind strongly to proteins bearing cationic sites.

Pasteless Biopotential Electrodes

Hard anodization Super has been authorized as a coating for aluminum kitchen utensils, and it proves to be very stable even under high temperatures and the presence of corrosive substances used while cooking. The coating does not wear off with the use of abrasive scrubbing pads and detergents. These properties indicate that no toxic substances are released in the presence of heat, alkaline or acid solutions, and organic solvents. This makes its use safe as a material in direct contact with skin, and resistant to sweat, body oils, and erosion due to skin friction.

Detergent Extraction Protocol

The following procedure is that of Manjunath et al. (18) with modifications described by Manjunath and Page (8) and Yeager (19). The yield is approx 0.4 mg of gap junction proteins per 1 g of heart. The two detergents used are Sarkosyl and deoxycholate. The protocol described here is for 4 rat hearts, but can be scaled up to 20 rat hearts (19).

Truncated Form of Cx43 from Stable Transfected BHK Cells

The major cardiac gap junction protein is Cx43. Expression of a truncated form of Cx43, Cx-263T, has been achieved in a stable transfected BHK cell line (14). The truncation occurs after Pro263 and removes most of the large C-terminal domain. The buffer used is HEPES, rather than the more commonly used bicarbonate and Tris buffers. The two detergents used are Tween-20 and DHPC. This protocol results in a much less pure fraction of gap junctions than can be obtained from either liver or heart tissue using the previously described protocols. The crystallinity of the purified gap junction plaques ( 7A), however, is the highest reported from all protocols and may be related to the deletion of a flexible cytoplasmic domain of Cx43. The following protocol is for confluent cell cultures grown on 20 T150 tissue culture flasks. See Chapter 4 in this volume.

Analysis of Cellular DNA Content by Flow Cytometry

Some methods utilize detergents and or hypotonic solutions to permeabilize cells (see Basic Protocol 2 and Alternate Protocol 2). Generally, this provides greater accuracy of the DNA content estimate compared to the measurement of fixed cells. The approach presented in Basic Protocol 2 combines detergent treatment with the use of proteolytic enzymes it is widely used for clinical material, especially for DNA analysis in samples of solid tumors. Alternate Protocol 2 is a simpler method, more applicable to uniform cell populations such as tissue culture cells.

Background Information

Exposure of live cells to detergents leads to rupture of the plasma membrane and leakage of cytoplasmic constituents isolated nuclei are then being measured. Since some cytoplasmic constituents are either autofluorescent, contain DNA (mitochondria), or may nonspecifically stain with DNA-fluorochromes, the accuracy of DNA content measurement for DNA ploidy or cell cycle phase estimate is greater when isolated nuclei rather than whole cells are analyzed. It should be stressed, however, that lysis of mitotic cells, which lack nuclear envelope, leads to dispersion of individual chromosomes or chromosome aggregates. The methods utilizing detergents or hypotonic solutions, particularly when the cell suspensions are mechanically agitated, pipetted, or vortexed, may therefore not detect mitotic cells. Furthermore, individual or aggregated chromosomes may be mistakenly identified as apoptotic cells with fractional DNA content ( sub-G1 cells). Lysis of apoptotic cells that have fragmented...

Hartreelowry Assay For Quantitation Of Total Protein

The original Lowry method for total protein analysis was first described in one of the most cited papers in biochemistry (Lowry et al., 1951). The assay is a colorimetric assay based on cupric ions and Folin-Ciocalteau reagent for phenolic groups. The assay has been reinvestigated many times and sometimes improved. Most of these studies were designed to discern how interfering compounds distort the assay and how detergents solubilize otherwise insoluble proteins. The literature related to this assay was comprehensively reviewed by Peterson (1983). This protocol describes Hartree's version of the Lowry assay (Hartree, 1972). This version uses three reagents instead of five, produces more intense color (increased sensitivity) with some proteins, maintains a linear response over a larger concentration range (30 to 40 greater), is less easy to overload, and overcomes the reagent salt coprecipitation encounter with Lowry reagents. Finally, the reagents formulation offers some advantages in...

Viral Hemorrhagic Fevers Vhfs

Viral hemorrhagic fevers are caused by a group of taxonomically different RNA viruses. All pose natural threats of infection, although the regions of where they are endemic may be confined to well-defined geographical territories. Under normal conditions, infection of humans occurs through contacts with infected animals or insect vectors. Four families, Arenaviri-dae, Bunyaviridae, Filoviridae, and Flaviviridae, although taxonomically diverse, are the primary causes of hemorrhagic fevers. The viruses are extremely pathogenic and, although relatively stable in aerosol form, are susceptible to detergents and household bleach. Their ability to replicate in cell cultures and their resulting yields make them potentially lethal as bioterrorist agents, particularly when introduced into ventilation systems.

Harvesting Of Protein Product From Batch Mode Spinner Cultures And Largescale Batch Reactors

Cell-associated product must be obtained by disrupting the cell wall and again separating the solid and liquid phase. Nonionic detergents may be used to weaken the membrane and aid the release of non-integral-membrane-associated material. Where the product is an integral membrane protein, the disrupted cell membrane should be recovered and a typical membrane protein extraction and purification protocol pursued (unit 4.2).

Biochemical Abnormalities

Abnormalities Shapes Lipids

A reduction of the effective intercellular lipid barrier properties can lead to deficiencies ranging from dry skin (depletion of lipids owing to excessive use of detergents), to hyperproliferation and abnormal scaling. Causes include essential fatty acid deficiency, abnormal intercellular deposition of various lipids, accumulation of

Strategic Planning Colorimetric Protein Assays

When confronted with the need to determine the total protein concentration of a sample, one of the first issues to consider is selection of a protein assay method. The choice among the available protein assays usually is made based upon consideration of the compatibility of the method with the samples to be assayed. The objective is to select a method that requires the least manipulation or pretreatment of the samples due to the presence of substances that may interfere. If the total protein concentration in the samples is high (i.e., in the range of 5 to 160 mg ml), the biuret total protein reagent is the best choice. If the total protein concentration in the samples is low (i.e., in the range of 1 to 2000 g ml), then any one of the other three (i.e., the Lowry, the Coomassie plus, or the BCA method) would be suitable. If the sample contains reducing agents or copper-chelating reagents, the Coomassie Plus Protein Assay Reagent (Pierce) would be the best choice. If the sample contains...

Preparing for Repeat Ion Exchange Step

Before repeating ion exchange, the solvent pH and ionic strength usually need adjustment. This can be carried out by dialysis (unit 6.2) or by gel filtration on a desalting column using, for example, Sephadex G-25 or G-50 (unit8.3). In preparation for cation-exchange chromatography, dialysis against slightly acidic buffers (pH 5.0 to 6.0) will result in the helpful precipitation of some E. coli proteins. It may also be advisable to include a relatively low concentration of urea (0.5 to 2 M) or a nonionic or zwitterionic detergent in the dialysis buffer to minimize coprecipitation with contaminants (for an extensive listing of detergents and properties, see Basic proteins (pi > 9.0), which do not bind to the DEAE column, can be

Preparation and Extraction of Insoluble Inclusion Body Proteins from Escherichia coli

Inclusion bodies recovered from cell lysates by low-speed centrifugation are heavily contaminated with E. coli cell wall and outer membrane components. The latter are largely removed by selective extraction with detergents and low concentrations of either urea or guanidine-HCl to produce so-called washed pellets. These basic steps result in a significant purification of the recombinant protein, which usually makes up 60 of the washed pellet protein. The challenge, therefore, is not to purify the recombinant-derived protein, but to solubilize it and then fold it into native and biologically active protein.

Affinity Chromatography Purification Of Gst Fusion Protein From Inclusion Bodies

In some cases, fusion proteins are entirely or primarily located in a denatured, aggregated form in inclusion bodies. Glutathione-S-transferase (GST) fusion proteins can often be purified from inclusion bodies after solubilization in urea or another denaturant followed by renaturation by dialysis (unit 6.3). Other methods of solubilization include addition of detergents such as Sarkosyl (N-laurylsarosine Grieco et al., 1992 Frangioni, 1992). After denaturation and renaturation, it is important to ensure that the protein has regained its native conformation and function (units 6.4 & 6.5). Although denatured GST will not bind to the glutathione column and hence will not be recovered by this method, the possibility that the GST moiety has refolded when the fusion partner has not folded properly should also be considered. As an alternative to purifying proteins from inclusion bodies, growing the cells at a lower temperature will often shift the fusion protein into the supernatant while...

Triton X100 Solubilization of Connexons

The first step in the biochemical detection of connexons by either chemical crosslinking or sucrose gradient velocity sedimentation is to solubilize these species from membranes in a manner that faithfully preserves their oligomeric state. In principle, this could be achieved by either dissociating fully assembled gap junctional plaques (see Fig. 1, no. 5) into individual connexons or by capturing nascent connexons (see Fig. 1, no. 2 and no. 3) prior to incorporation into junctional plaques. In the cell types we have examined, the first approach is of limited value. This is because most gap junctional plaques are exceptionally resistant to solubilization in common nondenaturing detergents, including Triton X-100 (TX-100), Triton X-114 (TX-114), octylpolyoxyethylene (POE), and octylglucoside (OG) when used in physiological salt solutions at 4 C (6). Harsher detergent treatments cause extensive breakdown of gap junctional plaques to individual connexin subunits rather than into connexon...

Sdspage Analysis of Connexons

After chemical crosslinking or sucrose gradient velocity sedimentation, monomeric and assembled connexin species must be immunoprecipitated and or analyzed by Western blotting using connexin-specific antibodies (11). Immunoprecipitants of connexins metabolically labeled with 35S methionine are detected after SDS-polyacrylamide gel electrophoresis by autoradiography or with a PhosphorImager. Unlabeled connexins are analyzed by immunoblotting either with or without prior immunoprecipitation (see Note 9). In the latter case, the presence of substantial amounts of TX-100 in the samples and their large volumes hinder analysis on minigels. We therefore precipitate the proteins in such samples using the method of Wessel and Flugge (12) prior to SDS-PAGE. This simple procedure yields near quantitative recovery of proteins and removes salts and detergents.

Removal of Contaminants

Lipids and other hydrophobic materials may be removed by washing with alcohol solutions (e.g., 70 ethanol) or nonionic detergents. When using alcohols or organic solvents to remove hydrophobic materials, it is often effective to wash first with a gradient from 0 to 100 and then with a gradient from 100 to 0 . Repeat washing with the alternating gradients until no contaminants are detected in the eluant. Precipitated materials. Precipitated materials that have accumulated in a column are very difficult to remove. Unless the precipitated material can be physically removed by removing the gel at the top of the column, the material will have to be resolubilized. Detergents, urea, and guanidineCl can be introduced to help dissolve contaminants. The viscosity of concentrated solutions used for cleaning may require very low flow rates. The column may be equilibrated and incubated in 6 M urea, then washed with distilled water and buffer. Degradative enzymes (e.g.,...

Commentary Background Information

The BCA protein assay is a modification of the Lowry protein assay in which enhanced color production is due to the reaction of reduced copper with bicinchoninic acid (BCA). This reagent has a unique advantage over the Lowry protein assay reagent and the Coomassie plus protein assay reagent because it is compatible with samples that contain up to 5 (v v) surfactants (detergents). Unlike the Lowry protein assay, all reactants needed are present in the BCA working reagent. Large numbers of Detergents Found that the anionic form of the Coomassie dye reacts primarily with arginine residues within the macromolecular protein. Coomassie dye reacts to a lesser extent with other basic amino acid residues (His, Lys) and aromatic residues (Try, Tyr, Phe) present in macromolecularproteins, but not with the free amino acids. Dye binding is attributed to van der Waals forces and hydrophobic interactions. The interference seen with bases, detergents, and other compounds can be explained by their...

Support Protocol 2

If detergents were used in the elution scheme, the regeneration protocol is more complicated. In this case the column should be washed sequentially with one column volume of distilled water one column volume each of 25 , 50 , and 95 ethanol two column volumes of -butanol one column volume each of 95 , 50 , and 25 ethanol, and finally one column volume of distilled water. This should be followed by reequilibration with the start buffer (see Support Protocol 1) or the storage buffer (see Storage of HIC columns, below).

Solubilization of Lymphocytes

The purification and study of transmembrane proteins requires isolation of these structures from their lipid environment. This isolation is carried out through the use of detergents. In this unit, several approaches to solubilization of membrane proteins are presented. Solubilization of whole lymphocytes, using conditions aimed at minimizing the disruption of protein-protein interactions, is described in the first basic protocol an optional step is included that may be useful when the disruption of protein interactions is desired as part of a purification protocol. In some situations, it may be desirable to purify membranes prior to their solubilization (see second basic and alternate protocols). To determine the physical relationship between proteins, a cross-linking protocol (see support protocol) is provided.

Preparation Of Membranes By Dounce Homogenization

Purification of membranes prior to their solubilization may be desirable for some experiments. This procedure is a significant purification step in that it removes cytosolic proteins and nuclei prior to solubilization of membrane proteins. Removal of nuclei prior to the addition of harsh detergents may allow increased solubilization of cytoskeletal-as-sociated proteins without the problems inherent in the solubilization of nuclei and subsequent release of chromatin.

Critical Parameters

This will help generate the active conformation and prevent coupling the lectin through the sugar-binding site. Also, keep the soluble lectin concentration below 10 mg ml during coupling or some may precipitate. Finally, do not couple free Ricinus communis II (RCA-II) from castor beans in your own lab because it is deadly the LD50 in mice is 0.8 J.g 100 g body weight. For all lectin column runs avoid high concentrations (> 1 ) of nonionic detergents or any SDS.

Critical Parameters and Troubleshooting

Second is the presence of nonionic detergents in the application solvent. Nonionic detergents are frequently used to solubilize intrinsic protein located in subcellular organelles such as membranes. These nonionic detergents form micelles that encapsulate immobilized dyes, making them inaccessible to proteins. Addition of a modest concentration of a negatively charged detergent, such as SDS or de-oxycholate, to an application solvent containing a nonionic detergent results in the formation of mixed micelles. The negatively charged immobilized dyes repel the negatively charged mixed micelles in solvents of modest ionic strength, preserving the accessibility of immo

Immunoprecipitation Using Cells Lysed Without Detergent

Immunoprecipitation of proteins that are already soluble within cells (e.g., cytosolic or luminal organellar proteins) may not require the use of detergents. Instead, cells can be mechanically disrupted by repeated passage through a needle, and soluble proteins can be separated from insoluble material by centrifugation. The following protocol describes lysis of cells in a PBS-based detergent-free lysis buffer. Other buffer formulations may be used for specific proteins.

Immunoprecipitation Using Cells Lysed With Detergent Under Denaturing Conditions

If epitopes of native proteins are not accessible to antibodies, or if the antigen cannot be extracted from the cell with nonionic detergents, cells should be solubilized under denaturing conditions. This protocol is based on that for nondenaturing conditions (see Basic Protocol 1, steps 1 to 7), with the following modifications. Denaturation is achieved by heating the cells in a denaturing lysis buffer that contains an ionic detergent such as SDS or Sarkosyl (N-lauroylsarcosine). The denaturing lysis buffer also contains DNase I to digest DNA released from the nucleus. Prior to immunoprecipitation, the denatured protein extract is diluted 10-fold with nondenaturing lysis buffer, which contains Triton X-100 this step protects the antigen-antibody interaction from interference by the ionic detergent. Immunoprecipitation is performed as described (see Basic Protocol 1).

Commonly Used Affinity Tags

Polyhistidine is such a ubiquitous affinity tag that most companies providing expression vectors or protein expression and purification reagents offer products related to this tag (see Table 9.9.1 for examples). Histidine readily coordinates with immobilized transition metal ions. Immobilized Co2+, Cu2+, Ni2+, Zn2+, Ca2+, and Fe3+ can all be used to purify polyhistidine fusion proteins, but Ni2+ is the most commonly used. Empirical determination of the most effective transition metal ion for purification of a specific polyhistidine fusion protein can be performed if purification by Ni2+ is unsatisfactory. There are several companies that offer IMAC resin. Iminodiacetic acid agarose (chelating Sepharose, Amersham Biosciences), nitrilotriacetic acid agarose (Ni-NTA resin, Qiagen), and carboxymethylaspartate agarose (Talon resin, Clontech) are all used for the immobilization of transition metal ions. Commercially available IMAC resins are unaffected by protease or nuclease activity, and...

Pilot Scale Freeze Drying

Selection and preparation of glassware and closures. Glassware (preferably type I glass formed from cylindrical tubes) should be washed without detergents and baked autoclaves to sterilize if low bioburden is required (see Note 12). To prepare such a protein it is most convenient to use small stoppered or screw-cap vials (usually glass) or to use ampoules that can be flame sealed after lyophilization. Closures could be siliconized to ease stoppering and should be baked to reduce residual moisture content (e.g., 16 h at 116 C) as otherwise this moisture may transfer to the lyophilized product on storage.

Preparing Tissue Culture Cell Extracts For Isoelectrofocusing

Preparation of samples for isoelectrofocusing containing relatively pure proteins is generally straightforward (see Basic Protocol 1, step 22). In contrast, complex samples such as whole-cell extracts, tissue extracts, or subcellular fractions are more difficult to prepare for successful isoelectrofocusing. Solubility limitations both prior to isoelectro-focusing and during focusing restrict analysis of these complex samples to protocols that include nonionic detergents and urea (see Basic Protocol 1). In addition, the presence of DNA and RNA in crude cell extracts further complicates isoelectrofocusing. The protocol presented below is suitable for preparing samples from cell cultures and is based on quantities compatible with silver staining or Coomassie blue staining. If smaller cell numbers and high-sensitivity detection methods such as autoradiography are used, volumes and quantities should be adjusted as needed.

Suspend 0510 x 106 cells in 50 l staining buffer in 12 x 75mm polystyrene tubes

The cells must be in a single-cell suspension so that they can be run in the flow cytometer. Many companies recommend lysing buffers to remove erythrocytes. Most of these lysing buffers contain detergents that permeabilize the cells and consequently they should not be used in this procedure. Erythrocytes can be removed by Ficoll hypaque discontinuous gradient centrifugation, or alternatively they can be lysed with VitaLyse (BioErgonomics), which does not contain detergent. Other means of removing erythrocytes without perme-abilizing nucleated cells, such as ammonium chloride lysis or QuickLysis (Cytognos), have not been assessed with this procedure but are likely to be compatible with EAS. The EAS procedure has not been successfully accomplished in the presence of erythrocytes.

External Disinfection of Dialysis Monitors

Materials of which the body of the machine and relative accessories are composed to avoid irreparable damage which could range from the simple opacity of the transparent panels to the actual 'melting' of some of the plastic components. On the whole, products containing benzene, acetone, toluene, xylene or similar solvents should be avoided. Finally external detergents and disinfectants should be used with disposable paper wipes (to be changed after use on each machine) avoiding every type of cross-contamination. Other critical components, possible causes of transmission of bacteria and or viral cross-infections from patient to patient, are constituted by the connection of the dialytic solution in the direction of and from the filter. Both the external and internal surfaces can be easily contaminated especially in the dismantling phase of the dialyzers, when the maneuver is carried out by operators wearing soiled gloves. Moreover, at the beginning of the dialytic session, before...

Biotinylation And Detection Of Cellsurface Proteins

Cell-surface proteins are labeled by a short incubation with an aqueous solution of sulfo-NHS-biotin. Unreacted biotin is quenched by incubation with serum-free culture medium, then cell membranes are solubilized with detergents and the lysates subjected to SDS-PAGE. The gel-resolved labeled proteins are transferred to nitrocellulose membranes by protein blotting. If desired, cell lysates can be immunoprecipitated prior to SDS-PAGE and blotting. Biotinylated proteins are then detected by reaction with strep-tavidin-conjugated alkaline phosphatase or horseradish peroxidase followed by visualization with an appropriate substrate.

Electroblotting from Polyacrylamide Gels

In some cases, stained blots are used only to identify protein band patterns while leaving the gel unmodified for subsequent steps (unit 10.8). If such minimal protein transfer is desired, contact blotting is a suitable alternative (unit 10.6). This unit also describes procedures for eluting proteins from membranes using detergents (Basic Protocol 2) or acidic extraction with organic solvents (Alternate Protocol 4).

Overview of Nucleic Acid Analysis

This chapter covers the methods most commonly used in analysis of nucleic acids. In unit 7.5, DNA analytical methods are presented that are adapted not only to DNA staining in fixed cells and in cells permeabilized by detergents, but also to supravital cell staining, staining of samples of cells isolated from archival material embedded in paraffin blocks, and detection of apoptotic cells characterized by fractional DNA content. A support protocol in unit 7.5 describes agarose gel electrophoresis of the degraded DNA, which is selectively extracted from the same apoptotic cells that can be identified by flow cytometry (Gong et al., 1994).

DNA Content Measurement for DNA Ploidy unit 75 and Cell Cycle Analysis

The second set of methods presented (see Basic Protocol 2 and Alternate Protocol 2) utilize detergents and or hypotonic solutions to permeabilize cells these methods generally provide more accurate estimates of DNA content compared to measurement of fixed cells. The approach presented in Basic Protocol 2 combines detergent treatment with use of proteolytic enzymes it is widely used for clinical material, especially for DNA analysis in samples of solid tumors. Alternate Protocol 2 is a simpler method designed for uniform populations (e.g., tissue culture cells).

Nonionic Detergent Solubilization Of Lymphocytes

Detergents are organic compounds that have both hydrophilic and hydrophobic characteristics. In their monomeric forms, they are soluble in both water and lipid. Choice of a particular detergent for initial solubilization or for addition after solubilization is somewhat empirical. Educated guesses can be made, however, as to the suitability of a detergent for a particular situation based on its structure and its intended use. Commonly used detergents and their properties are described in appendix 1. This protocol has been optimized for the solubilization of the T cell antigen receptor utilizing Triton X-100 as the detergent of choice. An optional step is presented in which the ionic detergents sodium deoxycholate (Na-DOC) and sodium dodecyl sulfate (SDS) are added after removal of insoluble material and nuclei. Addition of these reagents facilitates the disruption of interactions between integral membrane proteins and is useful for the purification of individual polypeptides. Their...

Sequencing Pvdfbound Samples Using A Blott Cartridge

For most applications where N-terminal sequence analysis of intact proteins or large peptides (> 6 kDa) is desired, the preferred final purification method is to use one-dimensional (unit 10.1) or two-dimensional (unit 10.4) gels followed by electroblotting to a high-retention PVDF membrane (unit 10.7) and staining with Amido black (unit 10.8). For more detailed discussion of these preliminary steps, see Strategic Planning. PVDF membranes have a high protein-binding capacity, bind large peptides and proteins with high affinity, and are inert to the harsh chemical environment in a protein sequencer. However, the wetability characteristics of these hydrophobic membranes are quite different from those of the hydrophilic glass filters (see Basic Protocol 1). These differences have necessitated development of specific sequencer programs and sample holders for PVDF membranes (see Fig. 11.10.2). The preferred method for loading proteins onto a PVDF membrane is by electroblotting from a...

Sequencing Liquid Samples On A Biphasic Cartridge Sequencer

The unique sample cartridge in the Hewlett-Packard sequencer (Fig. 11.10.2) results in quite different sample loading constraints than those encountered with sequencers utilizing a glass fiber filter (see Basic Protocol 1). Since samples are loaded onto the hydrophobic half of the cartridge unit, sample loading constraints are similar to those involved in applying a sample to a C18 reversed-phase column (also see unit 11.6). Large volumes of aqueous samples can be readily loaded. Most buffers, as well as high levels of nonvolatile salts that would severely interfere with sequence analysis on a glass filter, are easily accommodated because any hydrophilic buffers including those containing Tris and glycine can be readily washed out of the column. Because the binding of proteins or peptides to the sample cartridge is via hydrophobic interactions, only strong organic solvents or detergents are likely to interfere with sample binding. Samples in high concentrations of organic solvents can...

Disinfectants and Antiseptics

Disinfectants come from various chemical classes, including oxidants, halogens or halogen-releasing agents, alcohols, aldehydes, organic acids, phenols, cationic surfactants (detergents) and formerly also heavy metals. The basic mechanisms of action involve de-naturation of proteins, inhibition of enzymes, or a dehydration. Effects are dependent on concentration and contact time.

Isolation and Use of Rafts

Sphingolipid- and cholesterol-rich microdomains called rafts appear to exist in biological membranes, and can be isolated by their insolubility in the cold in nonionic detergents such as Triton X-100. Rafts are detergent insoluble because raft lipids are in an ordered state similar to the liquid-ordered (lo) phase described in model membranes. Rafts are important in immunology because the T cell receptor and several other important T cell-signaling proteins are present in rafts or are recruited there during signaling. Similarly, the high-affinity IgE receptor on basophils is recruited to rafts during signaling. In both cases, association of these proteins with rafts appears to be important in signaling.

Sample Preparation Guidelines

The detection limit for a peptide is dependent on the peptide's ionization efficiency, its mass, its hydrophobicity, its solubility, the availability of a basic site for protonation, and other unknown factors. MALDI is relatively tolerant of salts and small amounts of some detergents when compared to most other forms of mass spectrometry ionization (Mock and Sutton, 1992 Rosinke et al., 1995 Cohen and Chait, 1996 Gharahdaghi et al., 1996). Table 16.2.1 lists salt and detergent concentrations that are compatible with MALDI. Some of these components interfere with ionization, and others can prevent crystal formation. As a general rule, any component present in a concentration higher than that of the matrix ( 50 mM) may be a problem. Detection limits are quite dependent on sample purity, especially at

Sample Preparation for Maldi Mass Analysis of Peptides and Proteins

Sample preparation may be the most crucial step in mass spectrometric analysis of peptides and proteins by MALDI (also see sample preparation guidelines in unit 16.2). The free dipolar ionic peptides or proteins must be incorporated into a crystalline solid solution that is comprised of excess solvent (the matrix), which modulates desorp-tion ionization by preferentially absorbing the laser energy at the irradiation wavelength and laser pulse intensity being employed. Usually the cocrystalline sample and matrix are prepared by mixing solutions of each component and permitting the mixture to crystallize through evaporation of the solvents. Two methods employing this approach are presented in this unit the dried drop method (see Basic Protocol 1) and the rapid evaporation method (see Basic Protocol 2) also see Background Information and Table 16.3.1). This process can be disturbed by the presence of contaminants such as detergents and salts (see Table 16.2.1). Matrices that are...

Crystallization Procedures

The applied protein concentrations are rarely below 1 mgmL_ 1 or above 10mgmL_1 The pH ranges between 4 and 10 and is usually, but not always, fixed by a buffer. The most successful precipitants are polyethylene glycols with molecular masses of between 400 and 8000 that are chains of 9-180 oxyethylenes, at concentrations of 2-30 . Common detergents are compounds similar to octylglucoside and dodecylmaltoside as well as compounds similar to octyltetraoxyethylene and dodecyltetraoxyethylene. They are usually used at concentrations slightly above the critical micelle concentration (CMC). Detergents with higher polarity such as lauryldimethylaminoxide (LDAO) or even charged detergents such as dodecylsulfate turned out to be unsuccessful precipitants.50

Preparation and handling of cells

Cuvettes must be thoroughly and reproducibly cleaned by soaking in a proprietary cuvette-cleaning fluid such as Hellmanex II (Hellma), an alkaline liquid concentrate, or RBS-35 Detergent Concentrate (Pierce), following the manufacturer's instructions. The manufacturers of Hellma cuvettes caution against the use of ultrasonics as a means of boosting cleaning efficiency. Fifty percent nitric acid is also effective, but requires greater care and a fume hood. Detergents frequently contain fluorescent material and, if used, should be removed from cuvettes with 2 M HCl followed by water. After the final rinse with copious amounts of high-quality distilled water, the cells are best dried using suction through a Pasteur pipet protected with a small length of plastic tubing. This is safer than using acetone or alcohol, which can leach out fats from fingers and deposit them on the cuvette. The faces of cuvettes must not be touched after cleaning. Filling, emptying, and rinsing between...

Recent Studies on the Possible Identity of Microsomal CATa

Although CATA can be solubilized from rat liver microsomes using mild detergents such as deoxycholate5 and reconstituted into liposomes of known phospholipid compo-sition10 without loss of activity, further purification of the enzyme away from other membrane components requires harsher conditions, which inactivate the enzyme. For example, substantial further purification can be achieved by extraction into 8mM CHAPS with 4 M urea followed by anion-exchange chromatography and gel filtration (N. M. Broadway & E. D. Saggerson, unpublished work) but the protein then has to be detected by indirect means. Because of these difficulties, purification of microsomal CATa to homogeneity has not yet been achieved. It was during attempts to purify CATa, prior to sequencing, that we arrived at the notion that microsomal CATa and mitochondrial CFT, may be very similar proteins.

Combination Dnarna Fish and Immunophenotyping

In the last few years, several protocols have been developed allowing simultaneous detection of DNA or RNA sequences together with proteins. Before applying one of these protocols it is important to consider that DNA and RNA molecules differ in chemical nature and in localization within a cell. The consequence of this is that DNA and RNA FISH have different requirements concerning pretreatment of cells and hybridization conditions. Hybridization to DNA sequences requires denaturation of the target DNA, whereas RNA sequences are single-stranded. However, owing to the high structural complexity of many mRNAs, a controlled denaturation of target sequences often improves RNA hybridization signals (Dirks et al., 1993). The genomic DNA is tightly packed within the cell nucleus, so detection of a specific DNA sequence present somewhere within this genome requires measures to obtain full accessibility of the nucleus and removal of cellular components that may cause background signals....

Eucalyptus Eucalyptus spp Myrtaceae

Eucalyptus globulus subsp. globulus (the Tasmanian Blue Gum) and E. polybractea (the Blue-leaved mallee) are the two main species used for commercial medicinal production of the so-called eucapharma oils. The former is found particularly in Brazil, Argentina, Chile, Columbia, India, Russia, and Spain, where its oil is distilled from waste foliage from timber trees for use under the British Pharmacopoeia (BP) for coughs and chest conditions, as well as in household cleaning products and for balsam notes in perfumery. Eucalyptus polybractea oils are produced mainly in New South Wales and used in pharmaceuticals and dental products.

Indirect Agglutination Test

Indirect Agglutination Test

Tion assays are as follows massive dilution of the test sample addition of detergents covering of the bare surface of the sensitized particles with inactive proteins rigorous pretreatment of the test sample including heat treatment for 30 minutes at 56 C and enzymatic treatment with proteases reaction. These procedures are time consuming and can carry with them the undesirable effect of drastically reducing the potential sensitivity and accuracy of the immunoassay as a result of the required manipulations.

Peptide Sample Preparation by Ether Precipitation

This procedure is suitable for precipitating peptides from organic acids and is used routinely for concentrating synthetic peptides from the trifluoroacetic acid cleavage cocktail following 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis. It can be used to desalt peptides in nonvolatile buffers but is not recommended for removing detergents like SDS. Take appropriate safety precautions when using this technique ether is highly flammable, and a refrigerated centrifuge and fume hood are recommended. Practice the precipitation procedure with 5- to 25- g amounts of a standard peptide before attempting use with a precious sample. Maintain cold solution temperatures for best results.

Integral Membrane Proteins

High-resolution structural information is available for only 50 integral membrane proteins (Table 17.1.20), compared with over 10,000 soluble proteins, although an estimated 15 to 30 of genes encode membrane proteins. Difficulties in protein expression, solubi-lization, and the growth of well-ordered crystals have plagued structural studies. Nonetheless, remarkable progress has been made in solving structures of integral membrane proteins owing to advances in overexpression, availability of detergents, and the use of antibody fragments in crystallization. Complementary methodological approaches, including X-ray diffraction, cryo-electron microscopy, NMR spectroscopy, and electron paramagnetic resonance spectroscopy promise even faster future progress. Updated compendiums of structures of membrane proteins can be found at http blanco.biomol.uci.edu Membrane_ Proteins_xtal.html and http www. ct.html.

The Lowry Protein Assay For Determination Of Total Proteins

Lowry introduced this colorimetric total protein assay method. It offered a significant improvement over previous protein assays, and his paper became one of the most cited references in the life-science literature (Lowry, 1951). The Lowry assay is easy to perform, since the incubations are done at room temperature and the assay is sensitive enough to allow the detection of total protein in the low microgram per milliliter range. It is one of three copper chelation chemistry-based methods presented in this unit. Essentially, the Lowry protein assay is an enhanced biuret assay (see Basic Protocol 3). After a short incubation, Lowry's reagent C (Folin phenol) is added for enhanced color development (Fig. B1.1.1). The Lowry assay requires fresh (daily) preparation of two reagents and a meticulously timed incubation step. The two reagents are combined just before use to make a buffered alkaline cupric sulfate working solution. The addition of sodium dodecyl sulfate...

A Poly Acrylamide Gel Electrophoresis PAGE

While IEF is a powerful tool used primarily for confirming the identity of a protein biopharmaceutical, upon suitable validation it can also be used as a stability-indicating method to monitor changes to the protein over time 32, 33 , Isoelectric focusing is normally run in a native gel using wide pore polyacrylamide or agarose but the addition of nonionic detergents, nonionic chaotropic agents or reducing agents during sample preparation may serve to dissociate molecular complexes and aggregates resulting in enhanced resolution.

Direct Extraction Of Eicosanoids Into Organic Solvents

Extraction and purification of eicosanoids are likely to be necessary if there are contaminating substances in the sample that will interfere with the assay. Organic solvents, detergents, proteins, and high levels of salts can all interfere with obtaining good results. It is important to note that extraction procedures should be avoided, if possible, since they require substantial effort and always result in sample loss (see Critical Parameters). This protocol describes an acetone chloroform extraction method for extracting eicosanoids from samples. An alternate extraction procedure, which uses reversed-phase liquid chromatography is also described (see Support Protocol 2).

Measurement of MHCPeptide Interactions by Gel Filtration

This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to MHC molecules. MHC molecules are solubilized with detergents and purified by affinity chromatography. They are then incubated for 2 days at room temperature with the inhibitor peptide and an excess of a radiolabeled probe peptide, in the presence of a cocktail of protease inhibitors. At the end of the incubation period, MHC-peptide complexes are separated from unbound radiolabeled peptide by size-exclusion gel-filtration chromatography, and the percent bound radioactivity is determined. The binding affinity of a particular peptide for an MHC molecule may be determined by co-incubation of various doses of unlabeled competitor peptide with the MHC molecules and labeled probe peptide. The concentration...

Preparing Whole Cell Extracts

The second step in a successful coprecipitation is generating whole-cell extracts that optimize the yield and activity of the proteins to be analyzed, using lysis buffer conditions that permit recognition of the proteins by the affinity matrix. The yield of total protein in a whole-cell extract is not always a reliable indicator of the relative yield and activity of specific proteins, so it is wise to verify both parameters at the onset of an experiment before proceeding with the coprecipitation. Yield and activity can be affected by a number of factors (see Chapter 10 see Harlow and Lane, 1988). Small variations in the relative amounts of salt and detergents in the lysis buffer can have large effects on yield and activity, as can the speed and efficiency of cell breakage. Both factors are particularly important for less soluble proteins that associate with macromolecular structures such as membranes or cytoskeleton. In addition, global inhibition of proteolysis through the inclusion...

Design of Neuraminidase Inhibitors against Influenza

There are three types of influenza virus as classified by their serological cross-reactivity with viral matrix proteins and soluble nucleoprotein (A, B, and C). Only types A and B are known to cause severe diseases to humans. Type B is only found in humans, while type A occurs naturally in birds and mammals such as pigs and horses. Influenza, an orthomyxovirus, is a 100-nm lipid-enveloped virus. On the surface of the influenza virus there are two glycoproteins, hemagglutinin (HA) and NA, which appear as spikes protruding out of the viral envelope. There are between 50 and 100 NA spikes per virus.91 Electron microscopic images of the NA spikes reveal a mushroom-shaped molecule made up of a box-like head of about 80 x 80 x 40 A. It has a narrow centrally attached stalk (15A wide and 100 A long) which terminates in a hydrophobic knob anchored in the viral envelope.92 The spikes can be released by detergents and digested by pronase to release the NA 'heads,' which retain full antigenic...

Reagents and Solutions

Triton X-100 or Nonidet P-40 These are the most widely employed detergents for membrane extraction, although many available detergents can give a satisfactory result. Nonidet P-40 is no longer commercially available under that name but many companies still supply a chemically equivalent detergent. It is best to stay with a source that has worked in your own trials. We have had good results with Pierce's (Rockford, IL) Triton X-100 and ICN Biomedical's (Aurora, OH) Nonidet P-40 (Igepal CA-630).

Over Expression and Purification of Active unit 2111 Serine Proteases and Their Variants from Escherichia coli

To obtain the native, correctly folded, hence, active form of the protein from such aggregates, four steps are usually performed. (1) The bacterial cells are lysed and the aggregates, heavily contaminated with E. coli cell wall and outer membrane components, are collected by centrifugation. (2) The contaminants are largely removed by selective extraction with detergents, salts, and low concentrations of either urea or guanidini-umHCl to produce so-called washed pellets. (3) The aggregates are solubilized or extracted with strong protein denaturants such as guanidiniumHCl or urea and an oxido shuffling system based on oxidized (GSSG) and reduced glutathione (GSH). Extraction with a denaturant simultaneously dissociates protein-protein interactions and unfolds the protein. Redox buffers facilitate the later oxidation and refolding of the protein through thiol disulfide exchange reactions. Normally, GSSG GSH ratios of 5 1 to 10 1 are used, which are similar to those found in vivo in the...

Detecting HATagged Protein Based Probes Using AntiHA Immunoblot

The protein-based probes described here are not cell-permeable and can be used only to target active deubiquitinating enzymes (DUBs) that are either pure or present in cell extracts. For this purpose, cells are usually lysed using methodologies that do not involve detergents (glass beads, several cycles of freeze and thaw, or French press) in order to minimize interference with enzyme activity. More recent studies reveal that most DUBs retain their activity in detergent lysis protocols employing 0.1 NP-40. The amounts of probe and extracts used for labeling experiments need to be optimized depending on the nature of the biological sample. Usually, extracts prepared from cell lines contain a larger number of active DUBs as compared to extracts derived from tissue samples. The incubation times are also sample-dependent, but optimal labeling is typically observed after one hour at 37 C. HA-tagged probes can also be used to isolate and identify target peptidases by immunoprecipitation...

Preparing Protein Extracts for Quantitative Two Dimensional Gel Comparison

This unit provides methods for protein extraction and removal of interfering compounds for subsequent analysis of the proteins by two-dimensional (2-D) gel electrophoresis (unit 10.4). An important caveat is that these methods are intended only for two-dimensional separations in which the first dimension is isoelectric focusing (IEF). Thus, it is essential that the sample preparation process address the chemical constraints inherent in the IEF process and be reproducible between extractions to allow quantitative analysis of the resulting 2-D gels, the latter being crucial for quantitative proteomics analysis using 2-D electrophoresis (e.g., units 10.4 & 22.2). The procedures described in this unit address the first consideration by solubilizing the proteins in a solution similar to that used during IEF (i.e., a strongly denaturing solution of low ionic strength). The extraction solution usually contains high concentrations of urea and sometimes ancillary chaotropes such as...

Protein Extraction From Subcellular Organelles Other Than Nuclei

Subcellular organelles usually contain very low amounts of salts and nucleic acids, but can be very rich in membranes and thus in lipids and membrane proteins. In this case, incorporation of specific detergents in the protein extraction process can be very helpful to maximize the yield of the protein extraction. This combination of reducing agents and detergents prevents protein determination by most protein assays, however. Thus, the protein determination must be carried out on the organelle preparation prior to extraction.

Labeling Proteins With Acidcleavable Isotopecoded Affinity Tag Reagent

This procedure describes solubilization of proteins in isotope-coded affinity tag (ICAT) labeling buffer. If proteins are in a solution that is incompatible with labeling buffer ingredients, such as > 0.5 (w v) SDS or other detergents (see Critical Parameters), then the samples must first be cleaned up by trichloroacetic acid (TCA) precipitation (unit 3.4) or other compatible methods. The resulting pellet is resolubilized in the appropriate labeling buffer. It is sometimes necessary to first attempt solubilization of the pellet in a small volume of labeling buffer that contains a relatively high (1 to 2 w v) concentration of SDS. Once the pellet is solubilized, the solution can be diluted to reduce the SDS concentration.

Figure 2 Spectrophotometric detection of caspase activity Cleavage of the labeled colorless substrate DEVD by caspase

Classically, inorganic phosphate was determined as a complex of malachite green,10 and ammonium molybdate11 for determining the phosphatase activity of calcineurin. This method was further developed and ready-to-use reagents can be purchased (BiomolGreen from Biomol, Plymouth Meeting, PA, USA). This type of assay delivers highly reproducible results (Table 2). A disadvantage is the possible interaction of test compounds with the molybdate complex, resulting in false-positive compounds. This requires additional control experiments to exclude unspecific interactions. One of the easiest control experiments is running the assay in the presence of the potential active compounds in the absence or presence of inorganic phosphate without adding enzyme. In addition, secondary isotopic assays can further help to identify truly active compounds. In cases where inorganic phosphate is released by an enzyme reaction, this type of assay can be applied and is therefore not limited to phosphatases....

Cell Lysis For Protein Fractionation By Chromatofocusing Support Or Isoelectric Focusing Using The Rotoforprotocol

Cell lysis is a process that extracts proteins from cells. In order to keep extracted proteins intact in solution, the pH of the lysis buffer is controlled close to physiological pH using Tris buffer, and proteases are inhibited by protease inhibitors. Furthermore, various nonionic chaotropes, detergents, and reducing reagents can be used to enhance the solubility of proteins. In both the electrophoretic-based separation (liquid-phase IEF, Rotofor) and the ion-exchange-based separation (chromatofocusing), the ions will cause the focusing and protein binding, respectively, to deteriorate so maintaining low ionic strength is a key issue.

Polarographic Assay Of Diphenol Oxidases

This protocol is amenable to addressing several additional questions regarding DPO activity. Many plant o-DPOs are often present in a latent form (probably membrane-bound) and their activity may be enhanced in the presence of anionic detergents (chao-tropic agents). The measurement of latent DPOs using SDS is outlined below. Additionally, the assay can be used to study the effect of DPO inhibitors. Finally, the assay can be used to study kinetic parameters of DPO activity. The O2 electrode is the method of choice for the determination of enzyme kinetic parameters for DPOs since, unlike colorimetric assays, it measures the actual initial rate of O2 uptake. These parameters include Michaelis constants (KM) and inhibitor constants (K) for a full discussion of these aspects readers should refer to any standard textbook of biochemistry.

Method Specificity Selectivity

The FDA considers the removal of detergent residues an important aspect of the overall cleaning process 2 . If detergents are used to clean the manufacturing equipment, it is expected that they will be completely removed by rinsing, and that the removal be validated by testing for absence of residues. Unlike the situation with product residues it is not always as straightforward in selecting which component of the detergent to target for residue analysis. The pharmaceutical manufacturer may not have access to the specific composition of the detergent, although suppliers are usually willing to provide information on the chemical class of active agent(s). Moreover, there is commonly more than one component that is responsible for the cleaning action of the detergent product. It then becomes a question of whether to develop a test for a specific target component or for the whole detergent product. If choosing a single component, one should focus on testing for the particular component...

Spectrophotometric Measurement Of Conjugated Dienes

A recording double-beam UV spectrophotometer is used to determine the conjugated diene absorbance of product fatty acid hydroperoxide at 234 nm as a function of time. Although this method is used by a majority of investigators, it has limitations regarding (1) the clarity of the enzyme and substrate solutions, (2) UV-absorbing substances in the solutions, and (3) the presence of hydroperoxide-metabolizing enzymes that destroy the conjugated diene moiety. The method works best with purified LOXs, especially type 1 LOXs. With type 2 LOXs, it is especially important to use low substrate concentrations solubilized with nonionic, non-UV absorbing detergents, like Tween 20. Compared to the polarographic method, the UV method is over three-fold more sensitive over the measurable span utilized by the two methods (Figure C4.2.2).

Nonylphenol Ethoxylates PEGn Nonyl Phenyl Ether Polyoxyethylene n Nonyl Phenyl Ether

Their general formula is C9H19C6H4(OCH2CH2)nOH. Each non-oxynol is characterized by the number (n) of ethylene oxide units repeated in the chain for example, nonoxynol-9, nonoxynol-14. They are present in detergents, liquid soaps, emulsifiers for creams, fabric softeners, photographic paper additives, hair dyes, lubricating oils, spermicides, and anti-infective agents. They are irritants and sensitizers. Nonoxynol-6 was reported as a sensitizing agent in an industrial hand cleanser and in a crack-indicating fluid in the metal industry. Nonoxynol-9 is the most common

Optimized Fixation and Immunofluorescence Staining Methods for Dictyostelium Cells

Grafix Biochemical

Recent years have seen a powerful revival of fluorescence microscopy techniques, both to observe live cells and fixed objects. The limits of sensitivity, simultaneous detection of multiple chromophores, and spatial resolution have all been pushed to the extreme. Therefore, it is essential to improve in parallel the quality of the structural and antigenic preservation during fixation and immunostaining. Chemical fixations are broadly used but often lead to antigenicity loss and severe membrane damages, such as organelle vesiculation. They also must be followed by membrane permeabilization by detergents or solvents, which can lead to extensive extraction and cytosol leakage. Fixation with solvents bypasses the need for permeabilization, but when carried out at high temperatures, leads to severe extraction of soluble proteins and lipids and cytosol wash-out, and has therefore been used routinely to visualize the cytoskeleton. Here, we describe a few modifications to the common aldehyde...

Sizeexclusion Chromatography

ASephadex, Sephacryl and Sepharose are available from Pharmacia LKB. Bio-Gel A is available from Bio-Rad. Ultrogel AcA is available from IBF Biotechnics. Toyopearl is available from TosoHaas. *The fractionation range indicates the molecular size of a protein expected to elute at an elution volume equal to the bed volume (number on left) and the molecular size of a protein expected to be totally excluded from the column and to elute at the void volume (number on right). The fractionation ranges given are for dilute aqueous buffers and will differ for separations carried out under denaturing conditions (e.g., 6 M guanidineCl, 8 M urea, or detergents). aSephadex, Sephacryl and Sepharose are available from Pharmacia LKB. Bio-Gel A is available from Bio-Rad. Ultrogel AcA is available from IBF Biotechnics. Toyopearl is available from TosoHaas. *The fractionation range indicates the molecular size of a protein expected to elute at an elution volume equal to the bed volume (number on left)...

Cell Free Protein Expression

Vrrp Write

The described conventional in vivo technologies for protein production depend on the cellular integrity and are only suitable for the production of proteins that do not affect the physiology of the host cell.204,205 As already discussed, these methods are limited for the expression of many proteins, e.g., by the formation of inclusion bodies,206,207 by protein instability due to proteolysis,205,208 or by too low yields in case of most membrane proteins. High-level cell-free (CF) expression systems are a promising new tool for the preparative production of difficult proteins (Figure 2). CF systems are principally independent of the cell physiology and they allow direct and immediate control of the reaction at any time. A wide range of critical reaction parameters such as pH, redox potential, and ionic strength can be chosen and adjusted according to the requirements of the specific target protein. Furthermore, any additives that might help to stabilize the recombinant protein after...

Strategies For Isolation Of Insoluble Proteins

Recombinant proteins expressed in E. coli that are located in the low-speed pellet fraction (see Fig. 6.1.1) following cell lysis are highly aggregated (i.e., inclusion bodies). Inclusion bodies are normally derived from protein aggregation in the cytoplasm, or in the periplasm if a secretion vector was used. As mentioned above, protein can also be located in either the low- or high-speed pellet fractions because of interaction with bacterial nucleic acids. Furthermore, if the protein is known to undergo polymerization in vitro (e.g., viral nucleocapsid subunits), expression in E. coli can also be expected to lead to polymerization in vivo to varying degrees, and such proteins will be partitioned in both the supernatant and pellet fractions (Wingfield et al., 1995). There are also examples of membrane proteins that, when expressed in E. coli, associate with the inner cytoplasmic membrane and can be extracted with nondenaturing detergents (Bibi and Beja, 1994, and references cited...

Crystallization of Native Proteins

In simple terms, cells consist of a cytosol containing water-soluble proteins surrounded by a membrane filled with integral membrane proteins. Attempts to grow 2-dimensional crystals of membrane proteins within the membrane15 and to analyze them by electron diffraction using an electron microscope have been largely abandoned because of the limited rate of success. As an alternative, membrane proteins were crystallized in cubic and other lipid phases but this yielded only small crystals that were difficult to find and isolate from the highly viscous medium.16,17 Presumably, the viscosity reduces the diffusion rate too much for obtaining large crystals. In other experiments, the membrane proteins were removed from their natural environment by covering their nonpolar surfaces with detergents and then dissolving them in water.18 Such a soft micellar environment requires that the membrane protein is intrinsically stable so that it does not disintegrate when the natural pressure of the...

Effects of ETS on Respiratory Health

There exists a significant body of research on the potential effects on respiratory health from exposure to environmental (passive) tobacco smoke (ETS) (reviewed in National Academy of Sciences 2000). Many components of ETS are known lung irritants. There have been direct associations shown between exposure to tobacco smoke and the development of lung cancer, obstructive airway disease, chronic bronchitis, ear infections, and asthma. It is small wonder that ETS is associated with so many disparate disorders, considering that tobacco smoke components include the carcinogens benzene, toluene, and 1,3-butadiene (Mitacek et al. 2002), toxicants such as nickel (Tobacco Research Implementation Group 1998) and polycyclic aromatic hydrocarbons (PAHs) (Besaratinia et al. 2002), and common household chemicals including ammonia, formaldehyde, and acetone (Tobacco Research Implementation Group 1998). Moreover, the effects of tobacco smoke are not limited to the active smoker but to anyone exposed...

Figure

Economically, the most significant single use of proteases is their incorporation into detergents 7 . They facilitate the removal of biological dirt, which is mainly protein-based. Obviously, the characteristics of the proteases must be compatible with standard washing conditions. Accordingly, the most successful detergent proteases are stable at alkaline pH values at relatively high temperatures and in the presence of bleach, surfactants, and sequestering agents. Screening of proteases from a wide range of sources has identified members of the bacterial subtilisin subfamily as the most appropriate for detergent application 15,16 . Typically, they are optimally active at temperatures between 45 to 65oC and in the pH range of 9 to 12. Many such subtilisins have been modified by genetic engineering with the general aims of increasing their thermal stability as well as improving their resistance to oxidation.

Enzyme Engineering

The past two decades have witnessed intensive industrial and academic research attempts to develop techniques to engineer enzymes to meet industrial demands, to replace traditional chemical catalysts used widely throughout industry, and to broaden the scope of enzyme applications 9,14,15 . Perhaps subtilisin, a bacterial serine protease, best illustrates the multiple goals of enzyme engineering. Subtilisin has been used for many years as a key component of many detergents, in addition to being used in many other applications in industry. Thus far, mutations in over 50 of the 275 amino acid (aa) residues of subtilisin have been reported in the literature, resulting in improvements in subtilisin's overall catalytic activity, mechanism of action, stability (thermal and pH), substrate specificity, surface activity, folding mechanisms, and in the evolution of new activities for subtilisin (see 24 and references therein for a comprehensive review).

Summary

At present, representatives of the major MMPs have been successfully expressed in E. coli. Several expression systems have been utilized with or without the propeptide, including MMP catalytic domains. N and C-terminal fusion proteins with glutathione S-transferase, ubiquitin and p-galactosidase have also been also used. In the majority of cases, the expressed protein is found in IBs, even when the expressed MMP was fused to a soluble protein an exception was the yeast ubiquitin system, which yielded soluble protein. Most workers solubilize the IBs in urea, although guanidine and cationic detergents are also effective. Refolding has been successfully carried out using a variety of techniques, including stepwise lowering of denaturant through dialysis, refolding while bound to an insoluble matrix, and pulse dilution. However, there are relatively few reports of a detailed comparison between the recombinant and native proteins.

Protein folding

The folding of proteins to the native form is favored under physiological conditions. The native conformation is lost, as the result of denaturation, at extreme pH values, at high temperatures, and in the presence of organic solvents, detergents, and other denaturing substances, such as urea.

Cleaning

While a detailed treatment of cleaning supplies and methods is beyond the scope of this section, some discussion is warranted. Consult your microscope's manufacturer or local dealer for any booklets or written materials that might be available. Many service dealers can provide expert advice and tips, as well as proper cleaning supplies, so don't hesitate to consult them.

Localizing Protein

Apart from its use in dissecting the bacterial compartments, lysozyme is often employed to prepare complete cell lysates, especially in laboratories that do not have access to a French press. Cells treated with lysozyme can be disrupted with detergents or by brief sonication (unit 6.5).

Breaking Cells

Cells are efficiently broken by high-pressure homogenization using a continuous-fill French press, which is suitable for processing volumes of 40 to 250 ml (reviewed by Hopkins, 1991 see unit6.2). (Yeast cells can also be conveniently broken with the French press, although two passes are required). For volumes exceeding 500 ml, the Manton-Gaulin-APV homogenizer (APV Gaulin) should be used. Sonication is also useful for breaking cells but is best suited for volumes < 100 ml. Alternatively, the outer cell wall can be enzymatically digested with lysozyme (200 g ml) and the cells broken by detergents, sonication, or both (Kaback, 1971 Burgess and Jendrisak, 1975 unit 6.5). Proteins that are secreted into the periplasmic space can be selectively released by hypotonic (osmotic) shock (Heppel, 1967).

Membrane Proteins

Components of the erythrocyte membrane skeleton, for example, are peripheral membrane proteins. Although most peripheral proteins are removed by washing a sample with buffers, integral proteins cannot be removed by such treatments. To isolate integral membrane proteins, which are embedded within the lipid bilayer, one must use detergents that disrupt the bilayer and bind to the proteins, thus solubilizing them. In general, integral membrane proteins have a portion of their peptide sequence buried in the lipid bi-layer whereas peripheral proteins do not. However, the discovery of glycosylphosphatidyli-nositol (GPI)-linked membrane proteins added to the ambiguity of the situation. GPI-linked proteins are globular proteins with no membrane-associated peptide sequence, yet they require harsh conditions for solubilization.

Column Calibration

The influence of protein shape on the calibration curve may be eliminated by disruption of noncovalent bonds with denaturing solvents (e.g., guanidine hydrochloride). Disulfide bonds are broken by reductive cleavage with a reagent such as DTT, and reoxidation is prevented by carboxymethylation. This treatment converts the shape of sample and reference proteins to random coils. The physical size of a random coil is larger than that of a compact molecule of equal mass therefore a more porous gel is needed to separate the former species. Detergents may also be used to denature proteins. However, it must be noted that a protein-detergent complex is larger than the protein and that ionic detergents will introduce charged groups. Also, information about size or molecular mass obtained using detergent-denatured proteins may not be relevant to the native protein. As these types of treatment are frequently used to prepare proteins for analytical electrophoresis, the reader is referred to units...

Elution of Proteins

Elution of compounds bound to an immobilized hydrophobic ligand can be achieved in one of three ways (1) by a linear or stepwise decrease in the concentration of salt, (2) by addition of some proportion of organic solvent to the elution buffer, or (3) by addition of neutral detergents to the elution buffer. The most common way to desorb the proteins from the HIC support is to reduce the salt concentration of the buffer (see discussion above under Type and Concentration of Salt in Buffer), or to use water alone as an eluant. An alternative elution scheme for desorption of the bound protein may exploit the fact that low concentrations of water-miscible alcohols, detergents, and salting-in salts (i.e., chaotropic salts that decrease precipitation of hydrophobic compounds see Fig. 8.4.1) result in the weakening of the protein-ligand interaction. The nonpolar regions of the alcohols or detergents compete with the bound protein for the hydrophobic ligand, resulting in displacement of the...

Generality

Several applications are described below to demonstrate the wide possibilities of LATMAG that can be relevant to the quantitative detection of almost all biomolecules. Protocols are almost the same whatever the analyte to be assayed. Incubation of magnetic and latex biosensitized particles occurs in the presence of targeted molecules, in an appropriate buffer containing salts and nonionic detergents. Afterward, the test tube is placed in the strong magnetic field generated by a rare earth alloy. After magnetic sedimentation, the supernatant is removed. Dumbbells and free magnetic particles that cannot be physically separated at this stage are dispersed in the desired buffer, and observed by eye, analyzed by spectrophotometry to measure the absorbance, or deposited onto a glass slide that is placed under a microscope to determine the number of latex particles. Reference samples without target are always needed to evaluate the amount of nonspecific adsorption of magnetic particles onto...

Immunoprecipitation

Immunoprecipitation protocols consist of several stages (Fig. 8.3.1 see Basic Protocol 1). In stage 1, the antigen is solubilized by one of several techniques for lysing cells. Soluble and membrane-associated antigens can be released from cells grown either in suspension culture (see Basic Protocol 1) or as a monolayer on tissue culture dishes (see Alternate Protocol 1) with nondenaturing detergents. Alternatively, cells can be lysed under denaturing conditions (see Alternate Protocol 2). Soluble antigens can also be extracted by mechanical disruption of cells in the absence of detergents (see Alternate Protocol 3). All of these procedures are suitable for extracting antigens from animal cells. By contrast, yeast cells require disruption of their cell wall in order to allow extraction of the antigens (see Alternate Protocol 4).

Unit

Immunoprecipitation protocols consist of several stages (Fig. 9.8.1 see Basic Protocol 1). In stage 1, the antigen is solubilized by one of several techniques for lysing cells. Soluble and membrane-associated antigens can be released from cells grown either in suspension culture (see Basic Protocol 1) or as a monolayer on tissue culture dishes (see Alternate Protocol 1) with nondenaturing detergents. Alternatively, cells can be lysed under denaturing conditions (see Alternate Protocol 2). Soluble antigens can also be extracted by mechanical disruption of cells in the absence of detergents (see Alternate Protocol 3). All of these procedures are suitable for extracting antigens from animal cells. By contrast, yeast cells require disruption of their cell wall in order to allow extraction of the antigens (see Alternate Protocol 4).

Penile Disorders

Several types of inflammation problems may involve the penis and the urethra. Balanitis occurs when the glans, or head, of the penis becomes red and sore. Usually the cause is unknown, but it is sometimes caused by urinary tract infection or allergic reactions to clothing or detergents. In uncircumcised men, the irritation may result when the foreskin is narrow or difficult to retract, and secretions become trapped beneath the foreskin.

Biochemistry

Early research identified glucosylceramide as the compound that accumulated in Gaucher's cells but it was not until 1965 that the pioneering efforts of Brady, et al. 10,11 in the United States and Patrick 12 in the United Kingdom, revealed that a deficiency of the lysosomal enzyme P-glucocerebrosidase was the defect leading to the accumulation of glucosylceramide in Gaucher Disease. Following the identification of the defective enzyme, it was suggested, as early as 1966 13 that the disease may be treated by ERT. Several groups put a great deal of effort over the following years into purifying and characterizing the enzyme. Since we now know that P-glucocerebrosidase is hydrophobic in nature, unstable at neutral pH, and requires the presence of detergents for solubility and full activity 14-16 , it is not surprising that these early attempts to produce a pure preparation of the enzyme met with varying degrees of success 17-23 . In vitro studies have demonstrated that...

Sample Preparation

To obtain the most accurate PTC-AAA data, peptide and protein samples should be homogeneous (i.e., exhibit a single SDS-PAGE band and or a single N-terminal amino acid). In addition, the sample should be dissolved in a volatile solvent, free of salts and detergents. Common volatile solvents useful for transferring readily soluble samples to hydrolysis tubes include water (HPLC grade), aqueous 0.1 trifluoroacetic acid acetoni-trile, 30 to 50 methanol, ethanol, or acetonitrile, 0.1 to 1 N-ethylmorpholine acetate, pH 8.5, and dilute (0.1 ) HCl. Less soluble samples can be dissolved in 75 to neat organic acids such as formic, acetic, and trifluoroacetic acids. Whenever possible, enough sample should be prepared for duplicate analyses per hydrolysis (plan on 0.5 to 1.0 g per analysis). Useful data can be obtained with < 0.5 g of sample, but ubiquitous contamination makes routine analyses below this range more labor-intensive and difficult for any AAA procedure. Useful PTC-AAA data can be...

Proteins

Protein intake may also indirectly lead to the induction of adverse effects. A well-known example of interactions between food components resulting in the formation of toxic products is nitrosamine formation. Secondary amines from fish protein may react with nitrite, originating from vegetable intake resulting in the formation of nitrosamines (for nitrosamine formation, see Part 2, Chapter 9). If vitamin C is also a component of the diet, the formation of nitrosamines can be prevented. Vitamin C inhibits the nitrosation reaction.

Healthy Chemistry For Optimal Health

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