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Mon, 30 Apr 2018 | Protein Science

A 15-ml starting serum sample may have to be passed over this column several times to quantitatively deplete cross-reactivity. The serum may be analyzed after each or several passes, or an aliquot can be saved for analysis at a later time. 4. If cross-reactivity with homologous phosphoprotein(s) is anticipated, pass the flow-through through the homologous phosphopeptide affinity matrix column(s) using the methodology described in step 3. Refer to Strategic Planning for discussion of...

Prepare the pancreatic samples

Fri, 08 Dec 2017 | Protein Science

Remove pancreas from freshly sacrificed rat that has been fasted overnight (to maximize the zymogen granule population). Weigh pancreas (usually 0.8 to 1 g per Figure 4.2.1 Distribution of marker activities for various organelles after rate zonal centrifugation of a postnuclear supernatant (PNS) from rat pancreas using the conditions specified in Basic Protocol 1. The density profile of the gradient after the run was determined by refractive index and plotted as sucrose (sucrose plus Ficoll)...

Protein Elution From Pvdf Membranes Using Detergents

Thu, 16 Nov 2017 | Protein Science

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...

Transmembrane Protein Topology Types

Tue, 01 Aug 2017 | Protein Science
Transmembrane Protein Topology Types

Figure 1.5.2 Membrane proteins containing hydrophobic anchors. A nontransmembrane or monotopic membrane protein is anchored to the membrane via a hydrophobic amino acid sequence. Transmembrane proteins are classified as types I, II, III, and IV (Table 1.5.2). The first transmembrane segment of a multispanning membrane protein can be inserted as in type I, II, or III proteins. This segment functions as a start-transfer peptide. Subsequent transmembrane segments will function as stop-transfer and...

Selection And Preparation Of Dialysis Membrane

Wed, 14 Jun 2017 | Protein Science

Dialysis membranes are available in a number of thicknesses and pore sizes. Thicker membranes are tougher, but restrict solute flow and reach equilibrium more slowly. Pore size is defined by molecular weight cutoff (MWCO) i.e., the size of the smallest particle that cannot penetrate the membrane. The MWCO designation should be used only as a very rough guide if accurate MWCO information is required, it should be determined empirically (see Craig, 1967, for a discussion of parameters affecting...

Analysis of Membrane Proteins in Detergents

Wed, 14 Jun 2017 | Protein Science

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...

Analysis Of Proteins Using Nearuv Spectrophotometry

Wed, 12 Apr 2017 | Protein Science

This procedure identifies proteins using near-UV spectrophotometry. Protein solution is placed in a UV-transparent quartz cuvette, absorption of light is measured as a function of wavelength, and the resulting spectrum is transferred either to a displaying device (e.g., CRT or printer) for inspection or to a computer that can employ a spreadsheet or other program for derivative calculations and or quantitative analyses (see Support Protocol). Acid ethanol cleaning solution (see recipe) 4...

Selective Precipitation By Isoionic Precipitation Dialysis Method

Wed, 25 Jan 2017 | Protein Science
Dialysis Protocol Protein Pictures

One of the older means of rendering proteins salt-free or nearly salt-free (i.e., isoionic) is dialysis (also see unit 4.4 & appendix 3b). However, two problems frequently arise with conventional dialysis (1) when appreciable amounts of protein are present, osmotic effects result in swelling of dialysis bags as salt diffuses outward and (2) often it is quite uncertain where the isoionic point is, even if it is feasible to deionize by dialysis against a buffer. The resin deionization method...

Growth Of Mammalian Cells For Protein Expression

Wed, 07 Dec 2016 | Protein Science

Mammalian cell culture media are complex in comparison to the simple defined media used for propagation of bacteria and yeast. This complexity stems from the need to satisfy the many functions required for normal growth and metabolism of mammalian cells (Bettger and McKeehan, 1986). Nutrients must satisfy the requirements for growth, maintenance of the cell, and expression of products. Additional specific growth factors such as insulin or insulin-like growth factor (IGF) are usually required....

Gel Dialysis Of Protein Samples

Thu, 29 Sep 2016 | Protein Science
Dialysis Molecular Weight And Size

This protocol describes a simple method for dialyzing small interfering molecules and salts away from protein samples using an agarose or polyacrylamide gel (see Fig. 3.4.5 Freifelder and Better, 1982). Somewhat dense, well cross-linked gels are used to keep proteins from penetrating the gel while unwanted salts and low-molecular-weight compounds diffuse (are dialyzed away) into the gel. This is a micro to semimicro method, an alternative to conventional dialysis (Craig, 1967), and to the use...

Background Information

Mon, 09 May 2016 | Protein Science

Under nondenaturing conditions, in which protein activity, native charge, and conformation are sustained, electrophoretic separation depends on many factors, including size, shape, and charge. Characteristics such as intrinsic molecular weight (i.e., in the absence of denatu-ration), the number of isoforms, and the presence of multimeric proteins can be determined with nondenaturing electrophoresis (often called native electrophoresis). The most important application of nondenaturing...

Strategies For Isolation Of Insoluble Proteins

Mon, 01 Feb 2016 | Protein Science

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...

Desalting The Sample After Cysteine Modification

Mon, 19 Mar 2012 | Protein Science

Except for the in situ method Basic Protocol 9 , all of the protocols presented leave the protein in the presence of high salt and reaction by-products which are usually not compatible with subsequent analytical techniques. This problem may be remedied by one of the following desalting methods. The methods are presented in increasing order of technological complexity. Protein recovery is a concern because small quantities of protein may be lost in dialysis or gel filtration. Modern...

Protease Cleavage Of Fusion Protein In Solution To Remove Gst Affinity

Thu, 15 Mar 2012 | Protein Science
Uncleaved Thrombin Cleaved Protein

The glutathione-S-transferase GST affinity tag is removed by cleaving with thrombin pGEX-T vectors or factor Xa pGEX-X vectors . Conditions for optimal cleavage of each recombinant fusion protein must be empirically determined. Some of the parameters that can be varied include temperature, enzyme-to-substrate ratio, length of incubation, and buffer conditions. Proteolysis can usually be performed in the glutathione buffer used to elute the fusion protein from the column, either with or without...

Digestion Of Proteins In The Presence Of Urea Or Guanidinehcl

Tue, 13 Mar 2012 | Protein Science

Because many proteins are resistant to protease digestion in their native state, the majority of digests are carried out in the presence of urea and guanidineHCl. The highest concentrations of these chaotropes compatible with the activity of the most commonly used proteases are listed in Table 11.1.1. Including chaotropes in the digest mixture avoids having to denature or reduce and alkylate substrate proteins prior to digestion. This, in turn, eliminates the need to remove the denaturants by...

Folding And Purification Of Bovine Growth Hormone

Sun, 04 Mar 2012 | Protein Science

The high-level expression of bovine growth hormone BGH in E. coli results in the formation of highly aggregated protein inclusion bodies . To restore the BGH to its native, functional state, the E. coli cells are first lysed with the French press and the inclusion bodies are partially purified by selective extraction of soluble and particulate E. coli contaminants. The inclusion bodies are solubilized with guanidine-HCl to give protein that is unfolded and reduced i.e., with four free...

Phenylthiocarbamyl Amino Acid Analysis

Mon, 27 Feb 2012 | Protein Science

Phenylthiocarbamyl amino acid analysis PTC-AAA is an established method for picomole-level quantitative analysis based on the reaction of phenylisothiocyanate PITC with amino groups to form phenylthiocarbamyl amino acid derivatives, which are then separated by reversed-phase HPLC and identified and quantified by ultraviolet absor-bance. Since its introduction in the 1980s Koop et al., 1982 Heinrikson and Meredith, 1984 Tarr, 1986 , PTC-AAA has become the most popular precolumn-derivatization...

Graphical Determination Of Specific Productivity Of Cells Producing Heterologous Protein

Fri, 24 Feb 2012 | Protein Science

For definition of specific productivity, see Support Protocol 6. Materials Spreadsheet software with graphical output e.g., Microsoft Excel or Lotus 123 1. Sample cultures at time points 1 and 2, count viable cells, and determine protein concentrations see Support Protocol 6, steps 1 and 2 . 2. Plot the viable cell densities versus time and plot the protein concentrations versus time on x-y formatted charts using spreadsheet software capable of graphical output. 3. Curve-fit the charts with the...

Measurement Of Dynamic Column Capacity And Breakthrough Capacity Of Ionexchange Columns

Tue, 14 Feb 2012 | Protein Science

For chromatographic separations that will be performed on a routine basis as well as separations that will be scaled up, it is necessary first to optimize conditions with respect to resolution and to determine the capacity of the medium with respect to the target protein see Support Protocol 1 . Knowledge of the capacity allows optimal use of the gel medium in terms of cost and yield, and also allows working limits to be set on the sample load to ensure robustness of a particular purification...

Peptide Sample Preparation by Ether Precipitation

Wed, 08 Feb 2012 | Protein Science | 1 comment

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...

Differential Scanning Calorimetry

Sat, 04 Feb 2012 | Protein Science

Before beginning calorimetry, an instrument baseline is established by a buffer buffer scan. If there are changes in the shape and or relative position of the buffer buffer scan, the cells are probably dirty see Support Protocol 4 for cleaning procedure . Protein solution, dialyzed unit4.4 amp appendix3.b and in dialysis bag see Critical Parameters Equilibration buffer buffer solution used for the last dialysis see Critical Parameters for discussion of buffer selection Spectrophotometer and...

F

Wed, 01 Feb 2012 | Protein Science

Figure 6.1.2 Localization of secreted and periplasmic proteins in E. coli. Periplasmic protein produced via a secretion vector can leak into the medium and be recovered by centrifugation supernatant, S1 or filtration. Washing cells with an isotonic solution such as lightly buffered 0.15 M NaCl or 0.25 M sucrose can also release protein S2 . The compartmentalized periplasmic proteins are released by isotonic shock treatment by directly suspending normal cell paste or plasmolyzed cell paste into...

Reagents And Solutions

Mon, 14 Nov 2011 | Protein Science | 1 comment

Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock solutions, see APPENDIX 2E for suppliers, see SUPPLIERS APPENDIX. 100 mM Tris - Cl, pH 7.8 pH as determined at 4 C 100 mM Tris - Cl, pH 7.8 pH as determined at 4 C 10 mM EDTA from 0.5 M stock 0.5 mM phenylmethylsulfonyl fluoride PMSF 5 mM benzamidine - HCl 780 mg liter Prepare immediately before use Purification of PMSF can be added as 2.5 ml liter of 0.2 M PMSF stock solution. 60 mM ethanolamineHCl, pH...

Osmotic Dehydration

Wed, 21 Sep 2011 | Protein Science

Although ultrafiltration is a highly effective procedure for concentrating relatively large volumes, it may be less effective and convenient with small volumes and high protein concentrations. Osmotic dialysis relies on the use of a high concentration of material outside a dialysis tube to draw water and small-molecule solutes from its contents by osmosis, leaving a more concentrated protein solution within the dialysis tubing. Either sucrose or polyethylene glycol PEG Mr 15,000 to 20,000 may...

Diafiltration Or Concentration By Tangentialflow Ultrafiltration

Tue, 06 Sep 2011 | Protein Science

The most rapid method for dialyzing large samples is the use of ultrafiltration membranes fabricated into hollow fibers or spiral-wound layers, or, as in this protocol, in the form of stacked plates. The sample is allowed to flow tangentially over these membranes with recirculation. The semipermeable nature of the membrane then permits water and materials with molecular weights that are substantially below the MWCO value to cross through the membrane surface relatively unimpeded retained...