Chelation for Coronary Heart Disease

Chelation Natural Miracle For Protecting Your Heart

Chelation therapy has been conclusively shown to be up to 82 Percent Effective at dissolving the plaque that blocks arteries! In the ebook Chelation Natural Miracle For Protecting Your Heart you'll discover: The frustrating reason many doctors are ignoring Edta chelationor even openly rejecting it. The deadly heart surgeries even the American Heart Association admits are unnecessary. The hidden signs and symptoms of heart attacks and strokes? Are you in danger right now?. The average number of years stripped away by heart and vessel disease. Can you get them back?. The newest set of risk factors for heart disease (they'll likely surprise you!). Shady government practices that protect Big Pharma and keep Edta chelation out of the public eye. How the Roman Empire could have been savedif only they'd known about Edta chelation. Why Edta chelation is guaranteed to be safeeven in extremely high doses. (It puts aspirin to shame!). The shocking truth about plaque in young childrenand how to keep your little ones safer. Why dentists, artists and welders need Edta chelationand whether your workplace is dangerous too. The differences between IV and oral chelationand which kind of Edta is right for you. Other forms of chelationand how these little-known treatments can dramatically boost your health.

Chelation Natural Miracle For Protecting Your Heart Summary


4.6 stars out of 11 votes

Contents: Ebook
Author: Michael Cutler, M.D.
Price: $19.95

My Chelation Natural Miracle For Protecting Your Heart Review

Highly Recommended

The very first point I want to make certain that Chelation Natural Miracle For definitely offers the greatest results.

Purchasing this book was one of the best decisions I have made, since it is worth every penny I invested on it. I highly recommend this to everyone out there.

Download Now

Chelation therapy

Adult Chelation Therapy Encephalopathic BAL IM first, followed by EDTA IV. BPb > 100 mcg dL Same. BPb > 70-100 mcg dL Succimer orally only. BPb < 70 mcg dL Reduce exposure, no treatment indicated chelation contraindicated secondary to increased BPb levels from mobilized bone stores. FIGURE 21.6 Metaphysical lead lines. Chelating Agents BAL (British anti-Lewisite, dimercaprol) IM only, developed in World War II as antidote for Lewisite (arsine) and mustard gases sulfur donor and nonspecific chelator of Pb, As, Cu. Side effects hemolysis in G-6-PD deficiency, nephrotoxic in acid urine, peanut allergy give prior to EDTA for lead encephalopathy. CaNa2EDTA (calcium disodium ethylenedi-amine tetraacetic acid) IV > IM, water-soluble nonspecific chelator of Pb, Hg, Cu, Zn. Side effects nephrotoxic with oliguria, IM subcutaneous calcinosis, transient increased ALT AST, redistribution of Pb from stomach to brain in encephalopathy. Pediatric Chelation Therapy Encephalopathic BAL IM...

Removal of Residual Mucus and Epithelial Cells

Remove the mucosal strips and the stirring bar, rinse them in a Petri dish with fresh HBSS and transfer them to a new container with 100 mL of HBSS, 1 mM EDTA, pH 7.2. Stir at room temperature for 60 min to releases epithelial cells from the basal lamina. The solution will become cloudy as the epithelial cells detach from the lamina propria. Stirring must be gentle, yet vigorous enough to keep all tissue floating in suspension, and not simply to push the strip around at the bottom on the container. 4. Collect EDTA solutions in 50 mL centrifuge tubes. Spin down 470g for 5 min and resuspend in 15 mL RPMI media.

Biuret Assay For Quantitation Of Total Protein

Biuret assays are the oldest and least sensitive assays for total protein among the colorimetric (spectrophotometric) methods. Nevertheless, the biuret assay is still frequently used because it is easy to perform, reagents are easily prepared, and the assay is markedly less susceptible to chemical interference than other copper-based assays. The assay is based on polypeptide chelation of cupric ion (colored chelate) in strong alkali. Catalog listings for many commercially available proteins and enzymes include their specific activities based on biuret protein (mg-biuret-protein-1). In general, biuret assays are useful for samples containing 1 to 10 mg protein ml, which is diluted 5-fold by the added reagent to give a concentration of 0.2 to 2 mg ml final assay volume (f.a.v.). Most proteins produce a deep purple color, with maximum absorbance (Xmax) at 550 nm.

Bicinchinonic Acid Bca Assay For Quantitation Of Total Protein

The bicinchinonic acid (BCA) assay for total protein is a spectophotometric assay based on the alkaline reduction of the cupric ion to the cuprous ion by the protein, followed by chelation and color development by the BCA reagent. Either a micro or a semimicro procedure, the latter generating a final assay volume of 2 or 3 ml, may be used the semimicro procedure is most frequently used.

Collect the gradients at 4C

Assay the gradient fractions for the activity of interest. For some assays it may be necessary to reduce the sucrose content and or concentrate the sample. If so, dilute fractions 2- to 3-fold with 0.3 M sucrose 5 mM MES 1 mM EDTA 0.2 xg ml DPPD and pellet 1 hr (or shorter if the volume is small) at 100,000 x g, 4 C.

Percoll gradient stock solutions

5 mM MOPS or MES buffer 1 mM EDTA No-Percoll solution (high sucrose) 2.15 M sucrose 35 mM MOPS or MES 7 mM EDTA Prepare solution from the following stock solutions 2.5 M sucrose (ultrapure e.g., ICN Biochemicals) 1 M 2-N-morpholinoethanesulfonic acid (MES), pH 6.5 1 M 3-N-morpholinopropanesulfonic acid (MOPS), pH 6.8 0.2 M EDTA, pH 7.0 and 0.4 mM DPPD (diphenyl-p-phenylenediamine Kodak) in ethanol. 2.5 M sucrose can be stored indefinitely at -20 C 1 M MES or MOPS and 0.2 M EDTA should be filtered using 0.22- m Millipore filter (or equivalent) and can then be stored indefinitely at 4 C and DPPD stock should be made fresh before use.

Rate zonal centrifugation gradient stock solutions

Prepare gradient solutions from the following stock solutions 2.5 M sucrose (ultrapure e.g., ICN Biochemicals) 50 Ficoll400 1 M 2-N-morpholinoethanesulfonic acid (MES), pH 6.5 0.2 M EDTA, pH 7.0 and 0.4 mg ml DPPD (diphenyl-p-phenylenediamine Kodak) in ethanol. Sucrose and Ficoll stocks are stored long-term at -20 C gradient solutions are used within two weeks of preparation and the DPPD stock is made fresh and used without storage.

Alternate Protocol

WEHI-3 is a loosely adherent murine macrophage cell line and should be harvested from subconfluent (< 80 confluent) cultures. These cultures are harvested by decanting the medium, washing the flask one time with sterile, room temperature, 0.02 EDTA solution (versene 1 5000), and then adding 20 ml of the EDTA solution at 4 C. Cells are dislodged by gentle scraping, and are transferred to a sterile tissue culture tube and washed two times with complete DMEM-10 medium. One aliquot of cells is then used to establish a new culture and the remaining cells can be used for determination of IFN-j activity. Each 150-cm2 flask should provide 2 x 107 cells.

Culture Of Monolayer Cells

Frozen cells are thawed and grown in appropriate complete medium containing twice the maintenance amount of serum (see below). When the cells are confluent, they are treated with trypsin EDTA, diluted, and maintained in appropriate complete medium containing 10 FBS (see Table 5.12.2). Trypsin EDTA 0.25 (w v) trypsin 0.02 (w v) EDTA, 37 C

Fluorescencebased Assay For Leukocyte Chemotaxis

Newer migration chambers are available that permit the assessment of chemotaxis in a 96-well format using fluorescently labeled cells (Baly et al., 1997). These special chambers (NeuroProbe) allow the placement of darkened 96-well plates within the apparatus. In these chambers, plate-size polycarbonate filters are utilized to separate the upper wells from the dilutions of chemokines placed in the lower wells. Once the migration period is finished, EDTA can be added to the upper wells to disengage migrating fluorescent cells from the filter, so the total florescence on the plate can be assessed using a microfluorometer. These newer methods not only permit the rapid qualitative assessment of cell migration, but also require fewer cells per well and permit the examination of several additional experimental groups per chamber. 20 M EDTA in PBS (appendix 2a)

Additional Assays To Assess Chemokine Bioactivity

Finally, another method for assessing chemotaxis uses a lactate dehydrogenase (LDH) assay to assess cell migration. In contrast to the fluorescense-based assay described in Alternate Protocol 2, this method does not require labeling with calcein AM. Leukocyte migration is performed in a similar fashion as for microchemotaxis assays, except that 24-well Transwell plates (Costar) with removable 3- to 5- m membrane inserts are used. After incubation, 20 M EDTA treatment of the membrane, centrifugation of the 24-well plate, and subsequent lysis of the cells in the plate, the number of migrating cells are determined using a colorimetric assay for LDH (see unit 3.16). This assay measures the conversion of a tetrazolium salt by LDH into red formazan, which can be quantitated using a microtiter plate reader at an absorbance setting of 490 nm.

Identification Electrophoresis

Buffer 1 3 M NaCl, 10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5 (Sigma-Aldrich). 28. Buffer 2 10 mM Tris, 1 mM EDTA, pH 8.0 (Sigma-Aldrich). 29. Buffer 3 80 (v v) formamide, 4 mM EDTA, 5 TAMRA- or ROX-tagged molecular size standard (Sigma-Aldrich).

Materials see Notes 35 21 Cell Culture

When replating cells for recordings of cell motility these were dislodged with 0.5 mg mL trypsin, 0.54 mM EDTA in a modified Puck's saline and seeded in 6-well tissue culture plates in DMEM containing 25 mM HEPES. Cells were incubated for 24-48 h (37 C, 5 CO2) before video recordings were initiated.

Procedures Assays and Normal Values

Klima Marrow Needle Puncture

Obviously, if a venous blood sample is to be taken for the purposes of other tests, or if an intravenous injection is going to be performed, the blood sample for hematological analysis can be taken from the same site. To do this, the blood is allowed to flow via an intravenous needle into a specially prepared (commercially available) EDTA-treated tube. The tube is filled to the 1-ml mark and then carefully shaken several times. The very small amount of EDTA in the tube prevents the blood from clotting, but can itself be safely ignored in the quantitative analysis. Up to 20 years ago, blood cells were counted by hand in an optical counting chamber. This method has now been almost completely abandoned in favor of automated counters that determine the number of erythrocytes by measuring the impedance or light dispersion of EDTA blood (1 ml), or heparinized capillary blood. Due to differences in the hematocrit, the value from a sample taken after (at least 15 minutes') standing or...

The Bicinchoninic Acid Bca For Determination Of Total Protein

Smith et al. (1985) introduced the bicinchoninic acid (BCA) protein assay reagent. In one sense, it is a modification of the Lowry protein assay reagent. The mechanism of color formation with protein for the BCA protein assay reagent is similar to that of the Lowry reagent, but there are several significant differences. The BCA protein assay reagent combines the reduction of Cu2+ to Cu+ by protein in an alkaline medium (i.e., the biuret reaction see Basic Protocol 3) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) by bicinchoninic acid. The purple-colored reaction product of this method is formed by the chelation of two molecules of BCA with one cuprous ion (Fig. B1.1.3). The BCA copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The primary advantage of the BCA protein assay reagent is that most surfactants, even if present in the sample at concentrations up to 5 (v v),...

Nucleic Acidprecipitating And Metachromatic Dyes

The physicochemical basis of the spectral difference in fluorescence emission after acri-dine orange staining has been elucidated (Dar-zynkiewicz et al., 1983). Ribosomal RNA stained with acridine orange at concentrations ranging from 4 to 20 M shows red emission and a strong increase in light scatter, whereas with DNA this phenomenon is observed with dye concentrations ranging from 10 to 50 M (Darzynkiewicz et al., 1983). To obtain the same effect in cells, higher dye concentrations have to be used. In the presence of EDTA the

Techniques For Neural Differentiation Of Human Ec And Es Cells

Another monolayer technique used in neural differentiation of human ES cells involves the use of the bone morphogenic protein (BMP) inhibitor Noggin Gerrard et al., 2005 . Several studies have now shown that addition of Noggin to human ES cell cultures results in derivation of both mature neural and glial lineages Pera et al., 2004 as well as neural progenitor lines Gerrard et al., 2005 . The method used involved growing human ES cells on Matrigel in conditioned medium before EDTA treatment and culture in specified neuralizing medium supplemented with Noggin. The neural progenitor cells were then maintained for several passages before being replated as single cells, and differentiation was induced by addition or removal of growth factors.

Gel filtration buffer

PBS (appendix 2e) or other buffer compatible with both the column and sample. Protease inhibitors (e.g., 1 mM EDTA, 0.15 mM phenylmethylsulfonyl fluoride) as well as a bacteriostat (0.05 sodium azide) may also be added to the buffer. A reducing agent (2-mercaptoethanol, dithiothreitol, or tris(2-carboxyethyl)phosphine hydrochloride TCEP Cl ) may also be included to prevent proteins containing reactive cysteines from forming disulfide linkages.

Removal of Contaminants

Contamination with metals may cause blue or gray discoloration at the top of an ion-exchange column. These metals can originate from impurities in buffer salts or water, or they can leach from metallic system components in contact with buffer solutions. Metal contaminants may usually be removed by treating the column with several column volumes of 10 mM HCl (i.e., pH 2) saturated with EDTA. Manufacturer's guidelines should be checked for pH stability of medium and column before exposure to mild or strong acid solutions.

Commentary Background Information

The modified Lowry protein assay reagent will form precipitates in the presence of surfactants or potassium ions. The problem of precipitation that is caused by the presence of potassium ions in the sample can sometimes be overcome by centrifuging the tube and reading the color in the supernatant. Most surfactants will cause precipitation of the reagent even at very low concentrations. One exception is sodium dodecyl sulfate (SDS), which is compatible with the reagent at concentrations up to 1 (w v) in the sample. Chelating agents interfere because they bind copper and thus prevent formation of the copper-peptide bond complex. Reducing agents and free thiols interfere, as they reduce the phosphotungstate-phospho-molybdate complex, immediately forming an intensely blue colored product upon their addition to the modified Lowry protein assay reagent. The primary advantage of the Coomassie plus protein assay is that it is generally compatible with most of the buffers and reagents found in...

Isolation of Human Intestinal Mucosal Mononuclear Cells

0.15 (w v) DTT HBSS (prepared fresh see recipe) 1 mM EDTA HBSS (diluted from 10 mM stock see recipe) Enzymatic digestive solution (see recipe) Complete RPMI-10 medium, supplemented (see recipe) Ficoll-Hypaque solution, density 1.076 to 1.078 g ml (unit 7.1) 30 Percoll solution (see recipe)

Collect and prepare samples

Perform BM aspiration in the posterior iliac spina, using a 14- to 8-G biopsy needle in order to obtain enough BM particles. Collect 1.5 to 2 ml of bone marrow using tubes containing tripotassium EDTA or heparin as anticoagulant. Pass the aspirate 2 or 3 times through a 25-G gauge needle in order to disaggregate the bone marrow particles.

Materials 21 Cell Cultures

Culture medium 100 mg L KNO3, 10 mg L (NH4)2HPO4, 10mg L MgSO4-7H2O, 4 mg L EDTA, 3.5 mg L FeSO4 7H2O, 50 Jg L H3BO4, 10 Jg L MnSO4-4H2O, 5 Jg L ZnSO4 7H2O, 5 Jg L Co(NO3)26H2O, 5 Jg L Na2MoO42H2O, 25 ng L CuS04 5H2O, 10 mL L of saturated CaSO4 solution, 30 mL L soil extract, for details see (29).

Biochemical Characterization of Human Collagenase3

The inclusion of human collagenase-3 in the collagenase subclass of MMPs was initially based on structural comparisons, since its deduced amino acid sequence contained a number of features characteristic of these enzymes. Subsequent studies performed with recombinant protein produced in different eukaryotic expression systems provided definitive support for the proposal that this novel human enzyme was a bona fide collagenase. In this regard, the first studies were performed with a recombinant protein produced in a vaccinia virus expression system.1 This protein was active against type I collagen as well as against synthetic peptides used for assaying vertebrate collagenases. In addition, its pro-teolytic activity was fully abolished by EDTA, a typical inhibitor of metalloproteinases.

Immunophenotyping Of Peripheral Blood Leukocytes

Whole blood is collected using a tube containing EDTA as the anticoagulant. Whereas a conventional blood draw is 7 to 10 ml or more, these protocols require only 500 l or less. The blood cells in suspension are stained with three monoclonal antibodies that are directly conjugated to different fluorochromes. The erythrocytes are lysed, and the sample is centrifuged and resuspended in PBS. The stained leukocytes are then mounted on conventional uncoated glass microscope slides and air dried. Cells are fixed in acetone and stained for nuclear DNA by incubation with 7-aminoactinomycin D (7-AAD). Analysis in the laser scanning cytometer (LSC) is then triggered on the nuclear fluorescence, and a variety of parameters are measured for the three surface protein-associated fluorescence emissions, DNA-associated fluorescence emission, and forward light scatter. To facilitate data interpretation, the slide is counterstained with hematoxylin and eosin (H & E). The morphology of the different...

Firing patterns of vasopressin neurons

Phasic firing is the result of intrinsic membrane properties. The bursts are not passive responses to a phasically patterned input, nor do they reflect spontaneous oscillations of membrane potential. Instead, the bursts are regenerative, in that the first few spikes of a burst trigger prolonged activity. The bursting depends on intracellu-lar Ca2+ blockade of Ca2+ entry or chelation of intracellular Ca2+ will block phasic firing. At the start of a burst, a small but long-lasting depolarising after-potential (DAP) follows each spike, and these DAP's summate, bringing the membrane potential close to the spike threshold. After the first few spikes, a depolarising plateau, reflecting a persistent inward current, sustains a burst (Figure 7.1B). The plateau can be viewed as an alternative state of the resting potential. When depolarised by about 10 mV from its normal resting potential of about -70mV, a vasopressin cell will tend to settle at a new, more depolarised resting (plateau)...

Critical Parameters and Troubleshooting

A clean blood draw and gentle handling of specimens are required to avoid spontaneous platelet activation. If agonists are being added during processing to study cell reactivity, platelet aggregation could occur and interfere with analysis. It is important, therefore, that the samples be left undisturbed during any activation steps, followed immediately by fixation. Even in the absence of added agonist, LPA will form relatively quickly once blood is drawn into anticoagulant therefore, the time to processing should be kept to a minimum (< 20 min) to avoid artifactual LPA formation. Large platelet-platelet aggregates can exhibit high light scatter properties, and can interfere with analysis. These can generally be gated out, however, by taking advantage of their high platelet-specific and low leukocyte-specific fluorescence. EDTA-anticoagulated whole blood should never be used for LPA analysis, as the EDTA can cause in vitro dissociation of platelets from leukocytes.

Selfassembly of enamel proteins

The distinction among the proteins of developing enamel was initially based on procedural criteria, in which it was found that the extraction of scrapings of developing bovine enamel matrix with guanidine hydro-chloride readily solubilized the amelogenin fraction while a 'mineral-bound' enamelin fraction was only solubilized by a subsequent extraction-demineraliza-tion of the residue with guanidine-EDTA (Termine et al., 1980). This procedural definition of these two matrix protein components has been widely used but, due to the incomplete nature of the separation procedure, may have led to some ambiguities. More recently other components of the enamel protein matrix have been described which may reasonably be attributed to the enamelin fraction e. g., 'ameloblastin' (Krebsbach et al., 1996), 'tuftelin or tuft' protein (Deutsch et al., 1991, 1995a,b) and 'ameloproteinase-I' (Moradian-Oldak et al., 1996b). An tentative approach to the organization this protein data is presented in the...

Preparation Of Peptide Affinity Column Using A Thiol Resin

Peptides conjugated to carrier proteins via a thiol group (i.e., Cys residue) may be coupled to affinity resin via chemistry similar to that used for carrier conjugation (unit9.2). Note, however, that the peptide must be reduced before using this protocol. To prevent reoxidation of the peptide, avoid aeration and perform coupling reactions rapidly in a buffer containing EDTA. After the peptide is coupled to the resin, Cys is added to block any remaining unreacted active sites. Finally, the resin is thoroughly washed and stored with a preservative.

Intracellular Ca2 concentration

In response to any spike activity, there is a large Ca2+ entry into oxytocin cells and vasopressin cells via several different voltage-gated Ca2+ channels. In addition, intracellular Ca2+ stores can be mobilised via second messenger pathways to give very large increases in intracellular Ca2+ concentration ( Ca2+ ,) (see 10, 11, 12, 13 ). The dynamics of Ca2+ change differ between the cell types, as they differently express Ca2+ binding protein calbindin 4 . Oxytocin cells contain more calbindin than vasopressin cells, allowing them a higher Ca2+ buffering capacity, which prevents generation of DAPs and therefore phasic firing. DAPs and phasic firing can be evoked in oxytocin cells by neutralising calbindin or by increasing Ca2+ ,'. Conversely, phasic firing neurons can be switched to continuous firing by introduction of exogenous calbindin or by chelation of intracellular Ca2+. The amplitude of DAPs depends on Ca2+ influx through voltage-dependent Ca2+ channels of L- and N-types, but...

Differential Staining Of Dna And Rna Of Unfixed Cells With Acridine Orange

This protocol describes the application of AO to differential staining of DNA and RNA. The cells to be stained with AO can be either prefixed in ethanol (see Alternate Protocol) or permeabilized with the nonionic detergent Triton X-100 as described here. The permeabilization is done at low pH in the presence of serum proteins. At low pH most histones dissociate from DNA, making DNA more accessible to AO and thereby improving stoichiometry and accuracy in DNA detection (Darzynkiewicz et al., 1984b). Following treatment with the permeabilizing solution the cells are stained with AO dissolved in phosphate-citric acid buffer. The high molarity and excess of the buffer neutralize the acid maintaining the low pH in the first step. In the presence of EDTA, AO at the proper concentration interacts with cellular RNA to form condensed complexes that luminesce red, with maximum emission above 630 nm. At the same time, interactions of AO with DNA result in green fluorescence. Thus this...

Cell Growth and Induction

For construction of a plasmid which allows IPTG-inducible expression of the soluble and self-sufficient P450 BM3, the cytochrome P450 gene was amplified from plasmid pT-USC1BM3 (10) by PCR (11). DNA cloning was performed by standard recombinant DNA methods (12) using a QiaPrep Spin Mini Prep Kit (Qiagen, Valencia, CA) for isolation of plasmid DNA and a QiaQuick kit (Qiagen) for purification of DNA fragments from Tris-Acetate EDTA (TAE) agarose gels. Restriction sites for cloning of the gene behind the strong and inducible double tac promoter of pCWOri (+) (13) by BamHI and EcoRI sites were introduced by this PCR (11). In order to be able to evolve the heme domain independently from the rest of the enzyme, a silent mutation was introduced to produce a SacI site, 130 bases upstream of the end of the heme domain. The QuickChange (Stratagene, La Jolla, CA) protocol was followed. The resulting expression plasmid pBM3BamSacEco (Fig. 2) allows directed evolution of the hydroxylase domain...

The Coomassie Dyebinding Bradford Assay For Determining Total Protein

Coomassie Protein

In 1976, Marion Bradford introduced the first Coomassie dye-based reagent for the rapid colorimetric detection and quantitation of total protein. The Coomassie dye (Bradford) protein assay reagents have the advantage of being compatible with most salts, solvents, buffers, thiols, reducing substances, and metal chelating agents encountered in protein samples.

Background Information

There are a number of different procedures for the preparation of genomic DNA. They all start with some form of cell lysis, followed by deproteinization and recovery of DNA. The main differences between various approaches lie in the extent of deproteinization and in molecular weight of the DNA produced. The isolation procedure described here is relatively brief and relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent SDS. Use of proteinase K for DNA purification was described by Gross-Bellard et al. (1972) and Enrietto et al. (1983). EDTA is included in the digestion buffer to inhibit DNases.

Activation of Procollagenase by Trypsin

Trypsin-induced activation of procollagenase involves an initial cleavage between Arg36 and Asn,37 immediately after the triplet of basic amino acids, K34RR (Fig. 6.1), generating a major intermediate form of 46 kDa.28 This intermediate form is then converted to a stable 42 kDa active enzyme by cleavage between Phe81 and Val.82 While formation of the 46 kDa intermediate form is not affected by metalloproteinase inhibitors, EDTA or o-phenanthroline, subsequent conversion to the 42 kDa active form is completely inhibited by these reagents.28 Moreover, the conversion of the intermediate form to the 42 kDa species does not require trypsin activity, cannot be blocked by serine protease inhibitors, and is independent of the initial procollagenase concentration.14,28 These lines of evidence suggest that the proteolytic cleavage responsible for production of the 42 kDa stable active enzyme species is an auto-catalytic reaction requiring initial activation by trypsin.

Standard Cleavage Reaction

0.5 M EDTA (optional) The reaction can also be stopped by chelating Mg++ with 0.5 l of 0.5 M EDTA (final concentration 12.5 mM). If the digested DNA is to be used in subsequent enzymatic reactions (e.g., ligation or filling-in reactions), addition of EDTA should be avoided. DNA may be purified from the reaction mixture by extraction with phenol and precipitation in ethanol (unit 10.1) or with GeneClean (Bio101).

Isolation Of cDna Clones Encoding Intracellular Antigens

One day after transfection with a cDNA library, COS cells are replated onto a poly-vinylidene film. The following day, the cells are fixed with methanol. Cells expressing the target protein are radiolabeled by incubation with the appropriate antibody, followed by incubation with radioactive protein A or anti-Fc-antibody (alternatively, a radiolabeled primary antibody may be used). Autoradiography is used to identify the location of positive cells. Plasmid DNA is recovered by cutting the polyvinylidene wrap into squares and incubating them in SDS EDTA and is then amplified by transforming E. coli. The COS cell transfection and screening procedure is repeated using plasmid DNA prepared from pools of bacterial colonies. Individual colonies from the pools enriched with the target gene are screened until the gene is clonally isolated. This procedure is summarized in Figure 10.18.2. 1 nonfat dry milk in PBS with and without monoclonal antibody (MAb) 1 nonfat dry milk containing 125I-labeled...

The Biuret Assay For Determining Total Protein

Biuret Reagent Standard Curve 540

All proteins are composed of amino acids joined by peptide bonds in a linear sequence. There are 20 naturally occurring amino acids found in proteins. The amino acids are joined to each other by peptide bonds formed by a condensation reaction that occurs between the terminal amine of one amino acid and the carboxyl end of the next. Peptides containing three or more amino acid residues will form a colored chelate complex with cupric ions in an alkaline environment containing sodium potassium tartrate. A similar colored chelate complex forms with the organic compound biuret (NH2-CO-NH-CO-NH2) and the cupric ion. The reaction in which a colored chelation complex is formed with peptide bonds in the presence of an alkaline cupric sulfate solution became known as the biuret reaction (Fig. B1.1.5). Thus, the biuret protein assay reagent gets its name from the above reaction even though it does not actually contain the organic compound biuret. Single amino acids or dipeptides do not give the...

Analysis of Viral Infection and Viral and Cellular DNA and Proteins by Flow Cytometry

Flow and static cytometry monitor the infection and quantitate the viral-cellular events, on intact, single, fixed cells, for DNA, RNA, and proteins. This protocol was developed for the study of mammalian viruses, utilizing SV40 as the prototype (Jacobberger et al., 1986 Lehman et al., 1988 Laffin and Lehman, 1994), and to correlate viral proteins (T antigen and the VPs), cellular and viral DNA, and cell cycle proteins, such as the cyclins, PCNA, Cdks, Cdk inhibitors, pRb and p53, as infection proceeded (Friedrich et al., 1993 Whalen et al., 1999 Lehman et al., 2000). Cells are cultured, harvested using trypsin-EDTA to obtain a single-cell suspension, and fixed. The cells are stained with a suitable monoclonal or polyclonal primary antibody, then labeled with a FITC-conjugated secondary antibody to the protein of interest and with propidium iodide (PI) for DNA. Positive and negative controls should always be included. A wide range of cells have been analyzed (see below). 10x...


NaCl, 1 Nonidet P-40 (NP-40) (or IPEGAL CA 6310 Sigma, St. Louis, MO), 0.5 sodium deoxycholate (Fisher Scientific, Fairlawn, NJ), 0.1 SDS (Sigma, St. Louis, MO), 2 mM EDTA (Sigma, St. Louis, MO), 1 mM NaVO4 (Sigma, St. Louis, MO), 50 mM NaF (Sigma, St. Louis, MO), 40 mM P-glycerophosphate (Sigma, St. Louis, MO), 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma, St. Louis, MO), 10 g mL of pepstatin A (Sigma, St. Louis, MO), 10 g mL of leupeptin (Sigma, St. Louis, MO), 10 g mL of aprotonin (Sigma, St. Louis, MO). The protocol requires 4 mL of buffer per six oocytes. 2. IP buffer 2 (for cell lysis under denaturing conditions) 50 mM Tris-HCl base, pH 7.6 (TRIZMA Sigma, St. Louis, MO), 1 SDS, 100 mM NaCl, 1 mM NaVO4, 6 mM EDTA, 50 mM NaF, 40 mM P-glycerophosphate, 10 g mL of leupeptin, 10 g mL of pepstatin A, 10 g mL of aprotonin, 1 mM PMSF. The protocol requires 800 L per six oocytes. 3. IP buffer 3 (for IP under denaturing conditions) 50 mM Tris-HCl base, pH 7.6 (TRIZMA Sigma, St. Louis,...

Classification of microbicides

A large number of microbicides for material protection can be characterized according to their mechanism of action as either electrophilically active, membrane-active, or able to form chelates. This subdivision is indeed a rough one, but nonetheless helpful, although it is not always possible to classify active ingredients according to this distinction completely. The first step in the interaction of microbicides with microorganisms can be described as a physical process which occurs between the cell surface and the active ingredient. As much of the chemistry and functional physiology of the microbe cell, including energy production, protein synthesis, and nutrient adsorption takes place at the cell membrane, microbicides on their way to the membranes come across a lot of sites where they can exhibit their antimicrobial efficacy. Subsequent to association then follows as a second step, transport across the cell membranes, if the chemical and physical properties of the active...

Resolution Of Inositol Phospholipids By Thinlayer Chromatography

1 potassium oxalate 2 mM EDTA 50 100 1 (v v v) chloroform methanol concentrated HCl 100 mM EDTA, pH 7.4 100 mM KCl Chloroform 1. Pre-run silica-gel plate overnight in 1 potassium oxalate 2 mM EDTA and allow to air dry. Just prior to use, heat-activate plates by heating for 30 min at 110 C. 6. Add 0.03 ml of 100 mM EDTA, pH 7.4, 0.1 ml of 100 mM KCl, 0.15 ml chloroform, 0.15 ml water, and vortex for several seconds. Microcentrifuge 2 min and remove the lower phase (containing the lipids).

Quantitation Of Protein Carbonyls Derivatized With Tritiated Sodium Borohydride

3 M Tris-Cl, pH 8.6 (appendix2e) 0.5 M EDTA, pH 8.0 (appendix 2e) 3H NaBH4 working solution (see recipe) 2 M HCl 1. Prepare a 1 ml reaction mixture containing 86 mM TrisCl, pH 8.6 (add from 3 M stock), 0.86 mM EDTA (add from 5 M stock), 50 mM tritiated sodium borohydride (add from working solution described in Reagents and Solutions), and 0.5 to 1.0 mg of protein. EDTA is included in the reaction mixture so that any potential carbonyl-independent labeling can be prevented.

Blood Processing and DNA Extraction

EDTA anticoagulated blood tubes filled with whole blood (Vacuette, Greiner Labortechnik, Frickenhausen, Germany). 3. TKM 1 buffer 10 mM Tris-HCl, pH 7.6, 10 mM KCl, 10 mM MgCl2, 2 mM EDTA pH 7.6. 4. TKM 2 buffer 10 mM Tris-HCl, pH 7.6, 10 mM KCl, 10 mM MgCl2, 2 mM EDTA, pH 7.6, 0.4 M NaCl. 9. Tris-EDTA, pH 8.0 (TE).

Preparation Of Factor Ddepleted Serum Materials

1 M HCl (appendix 2) 0.5 M EDTA (appendix 2) VBS MgEGTA buffer (see recipe) 2. Adjust the pH of 50 ml fresh ACD-plasma to 7.0 with 1 M HCl. Add 1.0 ml of 0.5 M EDTA (to 0.01 M). Apply the plasma to the column and wash it through with 500 ml VBS buffer. Collect 5- to 10-ml fractions and read the OD280 to determine the location of the first protein peak.

Removal of the Epidermis from Fish Scales and Dissociating Melanophores from the Dermis

For working with melanophores from Fundulus heteroclitus, a small salt water fish, we remove the epidermis by shaking the scales in a marine Ringer's that contains EDTA. For removing the epidermis from the African cichlid, Tilapia mossambica, we use a fresh water Ringer's containing dispase, which enzymatically digests the matrix connecting the epidermis to the dermis. Once the epidermis has been removed from the scales, the scale with attached dermis is maintained in fresh water Ringer's. All Ringer's solutions can be made in advance and stored at room temperature or at 4 C. 2. Marine Ringer's with EDTA Marine Ringer's with addition of 10 mM EDTA and omission of CaCl2.

Fragmentation Of IgG Using Preactivated Papain

Additional Materials (also see Basic Protocol 4) 10 mg IgG (unit2.7) in 2 to 5 ml PBS Acetate EDTA buffer (see recipe) 2 mg ml papain in acetate EDTA buffer 1. Dialyze 10 mg IgG in 2 to 5 ml PBS in acetate EDTA buffer (appendix 3h). 2. Determine the concentration at A280 with the acetate EDTA buffer as a blank. Papain requires a free sulfhydryl group for its catalytic activity. In the native crystalline form, this group is blocked and the enzyme exhibits extremely low proteolytic activity. Cysteine activates the group and activation is optimal in the presence of a heavy-metal binding agent such as EDTA. 4. Equilibrate a PD-10 column with 20 ml of acetate EDTA buffer (appendix 31). 5. Apply papain cysteine (from step 3) to the PD-10 column. Collect ten 1-ml fractions, eluting with acetate EDTA buffer.

Culture Of Phoenix Cell Lines group nolan ) Complete medium Dulbecco's modified Eagle medium (DMEM) containing 10 (v v) heat-inactivated FBS, 100 U ml penicillin, 100 U ml streptomycin, and 2 mM L-glutamine PBS without Ca2+ or Mg2+ (e.g., appendix 2) 0.05 (w v) trypsin 0.53 mM EDTA 3. Add 2 ml of 0.5 trypsin 0.53 mM EDTA and allow cells to detach for 5 min at room temperature. Sharply rap the flask with the flat of the hand until cells separate into a mostly single-cell suspension.

Removal of the Epidermis

Transfer scales into a 35 mm Petri dish containing 3 mL of marine Ringer's with EDTA solution and incubate at 30 C for 30-40 min with occasional shaking. It is best to observe the scales against a black background with a dissecting microscope so that the clouding of the epidermis can be easily seen, indicating that it can be easily removed.

Construction of a Prokaryotic Expression Vector for IE2p86

1X TA buffer 40 mM Tris-acetate, 2 mM EDTA, pH 8.0. For staining of nucleic acids ethidium bromide is added at a concentration of 0.5 g mL. 6. Low salt buffer 0.2 M NaCl, 20 mM Tris-HCl, pH 7.3, 1.0 mM EDTA. High salt buffer 1.0 M NaCl, 20 mM Tris-HCl, pH 7.3, 1.0 mM EDTA.

Isolation Of Monocytes By Adherence

L-glutamine and 50 g ml gentamycin 0.02 EDTA in PBS (appendix 2 low pyrogen and without Ca++ or Mg++) 4. Remove adherent monocytes by gently scraping with a plastic cell scraper or by incubating the cells in ice-cold 0.02 EDTA PBS solution for 10 min followed by firmly tapping the flask. Transfer cells to a 15-ml conical tube and centrifuge 10 min in GH-3.7 rotor at 1400 rpm (300 x g) to remove the EDTA PBS solution.

Choice of Anticoagulant

Although sodium citrate (a weak calcium chelator) is the most common anticoagulant used for platelet studies, others have been successfully used. EDTA (a strong calcium chelator) should be avoided, because it causes dissociation of the integrin aIIbp3 (GPIIb-IIIa) complex. Heparin should also be avoided because it binds to, and may activate, platelets. Nonchelating anticoagulants such as P-PACK (a direct thrombin inhibitor) may be preferable for the monitoring of GPIIb-IIIa antagonist therapy. The anticoagulants listed in Table 6.10.2 have reportedly been used in studies of platelets.

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...

Nase I Protection Analysis

TE buffer 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0. 9. 10X TBE buffer, stock solution 108 g Tris base, 55 g boric acid, 4 mL 0.5 M EDTA, pH 8.0, dissolve in 1 L H2O. 4. Stop buffer 20 mM EDTA, pH 8.0, 0.2 M NaCl, 50 g mL yeast tRNA, 0.5 mg mL proteinase K (add proteinase K immediately before use). 6. Sequencing gel-loading buffer 98 deionized formamide, 10 mM EDTA, pH 8.0, 0.025 xylene cyanol, 0.025 bromphenol blue.

Visualization With Luminescent Substrates

EMcKimm-Breschkin (1990) reported that if nitrocellulose filters are first treated with 1 dextran sulfate for 10 min in 10 mM citrate-EDTA (pH 5.0), TMB precipitates onto the membrane with a sensitivity much greater than 4CN or DAB, and equal to or better than that of BCIP NBT. fLumi-Phos 530 contains dioxetane phosphate, MgCl2, CTAB, and fluorescent enhancer in a pH 9.6 buffer.

Preparation of High Salt Buffers

As the use of ammonium sulfate in buffers for HIC is very common, preparation of such buffers requires some comment. Because ammonium sulfate can be contaminated with heavy metals, iron in particular, the best grade available (usually called enzyme grade) should be used. For the same reason it may be necessary to include micromolar amounts of EDTA in HIC buffers containing ammonium sulfate. Preparation of a saturated solution takes several hours therefore it is best to prepare the solution a day before chromatography is to be attempted. Concentrations of ammonium sulfate are expressed in terms of molarity or percent saturation. For use in a saturation table, the sample can be considered to be 0 saturated or 0 M salt.

Preparation Of Spiked Control Blood Samples

Peripheral blood from a healthy nonpregnant adult Alsever's solution (see recipe) Umbilical-cord or newborn blood Anticoagulant (e.g., EDTA)-containing vacutainer Additional reagents and equipment for collection of EDTA-anticoagulated blood and determining blood ABO grouping 7. From a healthy nonpregnant adult, obtain a fresh ABO-compatible whole blood sample, anticoagulated with EDTA, with normal hematology CBC parameters.

Sample Requirements and Handling

Although protein concentrations in the range of 5 to 40 Mg Ml are generally employed for Raman spectroscopy, concentrations as low as 1 Mg Ml have been reported (Dong et al., 1998). Sample volume requirements are also modest ( 1 Ml). For most purposes, the sample is sealed within a glass capillary. When NIR excitation wavelengths > 752 nm are used to excite the Raman spectrum, quartz capillaries are preferred to circumvent fluorescence interference from glass. Quartz capillaries are also required for UVRR spectroscopy. Salts, buffers, and chelating agents appropriate for specific Raman spectroscopy applications to proteins are described in the general literature. Monatomic cationic and anionic species should be employed whenever possible, because polyatomic species generate Raman bands that may interfere with those of the protein.

Disrupt epithelial and mucosal cells

After decanting the supernatant, incubate the pieces in 20 ml HBSS EDTA for 30 min in a 37 C water bath, manually shaking the tube regularly during the incubation to ensure the epithelial cells are disrupted from the mucosa. 7. Wash off the remaining EDTA with 40 ml HBSS, calcium and magnesium free. At room temperature the density differences of living cells increase. Clumping of cells can be prevented by the addition of 1 mM EDTA to the solution.

Preparing L929 Conditioned Medium

L929 cells secrete granulocyte macrophage colony stimulating factor (GM-CSF) these cells can be used to condition medium to support the growth of macrophages isolated from bone marrow preparations. L929 cells must be treated with trypsin-EDTA solution to passage them. The conditioned medium is an economical reagent for use in expansion of bone marrow macrophages. 0.05 (w v) trypsin 0.53 mM EDTA solution (Life Technologies) Complete DMEM-10 medium (appendix 2) 2. Add 3 ml 0.05 trypsin 0.53 mM EDTA solution and let stand for 5 min. With cap tightened, tap flask sharply to dislodge cells.

Increasing The Sensitivity Of Fluorescent Detection Of Cellbound Peptide Using A Biotinylated Antiavidin Antibody

Dulbecco's PBS (see recipe) containing 10 mM EDTA Trypsin EDTA (Life Technologies) 2a. Optional Incubate 10 min at 37 C in Dulbecco's PBS containing 10 mM EDTA, followed by vigorous pipetting. 2b. Optional Trypsinize cells by overlaying with trypsin EDTA and incubating 15 min at 37 C.

Mechanisms Of Toxic Injury

The antidotes for heavy metals are called chelating agents, a picturesque term invoking an image of lobster claws (chelae) grabbing hold of the metal. Such drugs are rich sources of the ligands to which metals readily bind, and these drugs are able to compete effectively for the metal against the endogenous tissue ligands.

Transjunctional Metabolites Table

While the donor cells are being labeled with Dil, suspend the receivers in 50 mL of medium. Wash each of the five plates once with 2 mL of PBS (2 mL per plate) and then with 2 mL of trypsin EDTA. Tilt the plate for 30 s and aspirate completely to remove as much PBS and trypsin as possible (see Note 5). Monitor the cells under a microscope. When the cells separate well from the plate and each other, tap the plates sharply against a hard surface to release the cells. Suspend the cells from each plate in 10 mL of medium by vigorously passing up and down a 10-mL pipet several times before transferring into a sterile 50-mL polypropylene tube with cap.

Yeast Colony Hybridization

1 M sorbitol 20 mM EDTA 50 mM DTT (prepare fresh) 1 M sorbitol 20 mM EDTA 0.5 M NaOH 2. Remove membrane from plate. Air dry briefly. Incubate 30 min on a sheet of Whatman 3MM paper saturated with 1 M sorbitol 20 mM EDTA 50 mM DTT. 3. Cut a piece of Whatman 3MM paper to fit inside a 100-mm petri dish. Place the paper disc in the dish and saturate with 100,000 U ml -glucuronidase diluted 1 500 in 1 M sorbitol 20 mM EDTA (2 l glucuronidase per ml of sorbitol EDTA to give 200 U ml final). Layer nylon membrane on dish, cover dish, and incubate up to 6 hr at 37 C until > 80 of the cells lack a cell wall. The extent of cell wall removal can be determined by removing a small quantity of cells from the filter to a drop of 1M sorbitol 20 mM EDTA on a microscope slide and observing directly with a phase-contrast microscope at > 60x magnification. Cells lacking cell wall are nonrefractile.

Preparing Whole Cell Extracts

EGTA ( 15 mM) is commonly included to chelate divalent metal ions that are essential for metalloprotease activity. Because EGTA also inhibits other metal-dependent enzymes, it may be omitted, combined with the addition of a needed metal ion, and or substituted with EDTA.

Protease inhibitor stock solutions

Isopropanol (extremely toxic, unstable in water). Cysteine protease inhibitor 12.5 mg ml N-ethylmaleimide (NEM) in isopropanol Metalloprotease inhibitors 100.0 mg ml tetrasodium EDTA in PBS, pH 7.2 (appendix 2), and 26 mg ml 1,10 phenanthroline monohydrate in isopropanol Aspartic acid protease inhibitor 5.0 mg ml pepstatin A in isopropanol Serine and cysteine protease inhibitors 20 mg ml Na-p-tosyl-L-lysine chlo-romethyl ketone (TLCK) in water (bad odor use in hood) and 20 mg ml N-to-syl-L-phenylalanine chloromethyl ketone (TPCK) in isopropanol

Substrate Library Screen for Napsin A Using an Unpurified Cellular Lysate

Wash with NaOAc buffer supplemented with 1 mM EDTA, 10 g mL protease inhibitors E64 (Boehringer Mannheim), and 170 ig mL PMSF. 8. To 200 mM NaOAc buffer, pH 4.0, 1 mM EDTA, 10 g mL protease inhibitors E64, and 170 g mL PMSF, add 500 L of a 0.4 mg protein mL extract of napsin A transfected HEK293 cells to a final volume of 2 mL and add this to the library.

Development and Initial Validation of a Chemical method

Requirements and numerous details which may have bearing on the project range of concentrations needed and a potential limit of quantitation (LOQ), matrix to be used, anticoagulants in the case of blood or plasma (e.g. EDTA - what concentration , heparin - which salt ), volume of blood needed or available per assay (pediatric studies and studies using small animals provide small sample volumes, usually < 0.5 mL), stability, and safety considerations. The stability considerations are of utmost importance. For new chemical entities (NCEs) introduced for the first time into animals the stability data in biological matrices are frequently not available and neither is the method. One must immediately develop a skeleton of an assay and employ it to evaluate potential stability problems. Having potential stability or instability issues at least partially resolved one must then make sure that appropriate sample collection and storage procedures are used in clinics or animal rooms.

Purification of Highly Purified High Uptake rhIDU

Purification of the harvested culture fluid begins with acidification of the load and filtration of the harvest fluid (Figure 12.4). The cleared supernatant is loaded onto Blue-Sepharose FF resin, washed, and eluted with high salt. The eluate is loaded onto a Cu2+ chelation Sepharose column, and eluted under acidic conditions with pH 3.7 and 10 glycerol. This step was found to reduce CHO cell proteins. The pH 3.7 eluate is held as a viral reduction step, then the pH and salt level are raised and the fraction loaded onto phenyl-Sepharose HP for the final chromatography step. The enzyme is finally diafiltrated and concentrated into a formulation buffer (100 mM sodium phosphate buffer, pH 5.8, containing 150 mM NaCl), passed through DNA and viral retentive filters, and then 0.001 polysorbate-80 is added. The overall yield from the purification is about 40 and provides an enzyme of 99 or greater purity.

Homologous Recombination of a Mutant Transgene

Time course of cleavage at the helical and aminotelopeptide sites in mouse type I collagen by purified rat interstitial collagenase and human fibroblast (MMP-1) collagenase. (A) SDS-PAGE (5 acrylamide) showing reaction products after incubation with + + (Lanes 1-5 and 11-15) or r r collagens (Lanes 6-10 and 16-20) and sufficient purified human fibroblast (MMP-1) collagenase (Lanes 1-10) or rat (lanes 11-20) interstitial collagenase to digest 50 of the pepsinized + + collagen after 24 hr at 20oC. The substrates were collagen extracted from skin of + + mice with pepsin and from skin of r r (Col1a1tm1Jae) mice in 0.5M acetic acid without pepsin as described. 6 Parent solutions containing enzyme and substrate were prepared at 0oC and incubated at 20oC to start the reaction. Aliquots calculated to contain 25 ig of collagen were withdrawn and the reaction stopped by the addition of 1 10 vol of 500mM EDTA at the indicated intervals. After the reactions were stopped by the addition...

Metalloprotease Inhibitors

Protease inhibitors are essential partners for the investigation of proteolytic enzymes. They are useful not only for characterization of proteases but also for inhibition of unwanted pro-teolysis in an experimental system. Inhibitors may also have therapeutic value. Chelating agents such as EDTA and 1,10-phenanthroline are commonly used to block metallopeptidase activities in in vitro experiments. Synthetic inhibitors have been designed to inhibit specific metalloproteases. They commonly contain a chelating moiety, such as a carboxyl, a thiol, a phosphorous, or a hydroxamic acid group. The chelating group is attached to a series of other groups that fit the specificity pocket of a particular metallopeptidase. Because unbalanced activities of metalloproteases are involved in a variety of diseases, they are considered good therapeutic targets. Captopril, a thiol compound, is a potent inhibitor of peptidyl dipeptidase A (angiotensin I converting enzyme), and is used to treat...

Characterization of Collagenase Cleavage

We have expressed in E. coli human MMP-1 collagenase, (HCL1 called CLH in ref. 56 ) and mouse interstitial collagenase (MMP-13 MCL3 or CLM ) cDNAs and chimeric constructs in pET-3d, juxtaposing mouse collagenase (MMP-13) sequences N-termi-nal to the Zn-binding site in the catalytic domain and MMP-1 sequences C-terminal (MCL3 HCL1 or CLMH) and vice versa (HCL1 MCL3 or CLHM).56 MMP-13 (MCL3 or CLM) and chimeric molecules that contained the MMP-13 sequences N-terminal to the Zn-binding site (MCL3 HCL1 or CLMH) cleaved (+ +) collagen at the helical locus and cleaved crosslinked (r r) collagen in the N-telopeptide (p components converted to a chains). Human MMP-1 (HCL1 or CLH) and chimeric MMP-1 MMP-13 with MMP-1 sequences N-terminal to the active site (HCL1 MCL3 or CLHM) cleaved collagen at the helical locus but not in the N-telopeptide. All activities were inhibited by TIMP-1, 1,10-phenanthroline and EDTA. Thus, sequences in the distal 2 3 of the catalytic domain appear to determine the...

Oxidation and Proteolysis

Oxidation and proteolysis were recognized as the main destroyers of axone-mal function since the detergent-extracted models were first produced in the Gibbons lab (5). DTT and EGTA, commonly used in most reactivation methods, are the ingredients that protect against these spoilers. DTT competes with protein as a sink for oxygen free radicals. EGTA chelates contaminating divalent cations to a level that interferes with calcium-dependent proteases. It also inhibits the creation of free radicals via the Fenton reactions (which require Fe3+ or Cu2+ ions). In this way, EGTA (and also EDTA) can mask a multitude of sins. If the reactivation method is clean, and the reagents and water are pure, good reactivations can be produced without EGTA or EDTA. We are in the habit of routinely including 0.5 mM EGTA. Hypothetically, if oxidation and enzymatic degradation could be stopped completely, then reactivated motility

Extraction and Quantitation of Total RNA

Guanidine isothiocyanate (GITC) solution 59 g GITC, 0.24 g Tris-HCl, 0.34 g ethylenediamine tetraacetic acid (EDTA), 50 mL sterile H2O, heated to 60 C to dissolve GITC, then cool, and dilute to 100 mL with H2O. Then filter through a 0.45- im nylon filter into a sterile bottle. Add 5 mL 2-mercaptoethanol (Fisher, cat. no. BP176-100) prior to use. 2. Cesium chloride (CsCl) solution 96 g CsCl, 1.68 g EDTA, and 50 mL sterile H2O. Then autoclave and use H2O to adjust the final volume to 100 mL.

Strategic Planning

Like a metal chelator (such as 1 mM EDTA) or a reducing agent (such as 1 mM dithiothreitol). It is important to make sure that all components included in the extraction buffer are compatible with each phase of the assay. A sample protocol for enzyme preparation from kiwi fruit is presented in the Support Protocol.

Materials And Methods

Synthesis of 1-14C acetylcarnitine l-14C Acetylcarnitine was prepared enzymati-cally from 1-14C acetyl-CoA in an incubation mixture (final volume 1ml) containing 25mM HEPES pH 7.6, 2mM N-ethylmaleimide (NEM), lOmM EDTA, 40 pM carnitine (Sigma Chemical Co., St. Louis, MO), 44 pM (600,000dpm) l-14C acetyl-CoA (Amersham Life Science) and 50 (xg (4U) carnitine acetyltransferase (Boehringer Mannheim). The reaction was allowed to proceed for 1 hour at 25 C. Thereafter excess N-ethylmaleimide was neutralised by addition of 10pi 200 mM cysteine. The reaction mixture was applied to a Dowex AG-1-X8 anion exchanger (1ml bed volume, Cl-form, 200-400 mesh) to remove unreacted l-14C acetyl-CoA from the reaction mixture (recovery typically > 90 ). The elvant was used directly as substrate for activity measurements. Carnitine acylcarnitine carrier activity measurements The reaction was performed in a glass tube with a rubber septum. This tube contained two smaller tubes, one containing the reaction...

Intracutaneous injection and recovery of cervical lymph nodes

Most assays that follow immunological aspects of the infection focus on gut-associated lymphoid tissues (GALT), including the mesenteric lymph nodes draining the site of adult worm infection, the Peyer's patch in the duodenum and jejunum, and cells that can be released from the intestinal epithelium and lamina propria using EDTA and enzymatic dissociation methods. The tracheal bronchial lymph nodes have also been isolated to characterize responses from the draining lymph nodes of the lung following migration of developing L4. Early development of infective to parasitic L3 in the skin can be assessed immunologically by removal of cervical lymph nodes that drain the ear when L3 are injected intracutaneously.

Establishing The Primary 4t1 Tumor

4T1 tumor cells (ATCC also available from Fred Miller, University of Michigan) IMDM, DMEM, or RPMI with and without 10 (w v) FBS (appendix 2a) 100x antibiotic-antimycotic (Life Technologies) 0.25 trypsin 1 mM EDTA (Life Technologies) 8-week-old female BALB c mice (The Jackson Laboratory)

For microplate format

In an appropriate number of wells in a 96-well microplate, mix 10 l of 2 to 2000 nM MMP solution with 90 l TNC assay buffer. As negative controls, include one set of wells with 10 l TNC assay buffer instead of enzyme, and another with 10 l MMP solution, 2 l of 0.5 M EDTA (10 mM final), and 88 l TNC assay buffer. Equilibrate 10 min at 37 C. Stop solution contains EDTA which stops the reaction by chelating the Zn2+ required for MMP activity. 1b. To each microcentrifuge tube, mix 50 l of 2 to 2000 nM MMP solution with 450 l TNC assay buffer. As negative controls, include one set of wells with 50 l TNC assay buffer instead of enzyme, and another with 50 l MMP solution, 10 l of 0.5 M EDTA, and 440 l TNC assay buffer. Equilibrate 10 min at 37 C.

Preparation of S17 Feeder Layer

Mouse bone marrow S17 cells (3) are maintained in S17 culture medium. To prepare feeder layers, the S17 cells are dissociated with Trypsin-EDTA and irradiated (20 Gy see Note 7 ). Irradiated S17 cells (2.5 mL at 1.0 x 105 cells mL are plated onto 0.1 gelatin-coated six-well plates (thus, 2.5 x 105 cells per well). Feeder layers should be prepared 1 or more days prior to co-culture with hES cells and remain suitable for use up to 2 wk when kept in a 37 C, 5 CO2 incubator.

Dissociation and Harvesting of Differentiated hES Cells

Remove the supernatant and add 1.5 mL of trypsin EDTA + 2 chick serum solution into the tube. Warm at 37 C in a water bath for 5 to 15 min. Vigorously vortex and observe samples at 3- to 5-min intervals until there are few, if any, clumps of undispersed cells. 3. Add 6 mL of DMEM containing 10 FBS (D-10 medium) to neutralize the trypsin-EDTA and pipet up and down to further disperse cells. Centrifuge at 400g for 5 min. Resuspend cell pellet with 5 mL of D-10 medium. Filter the cell suspension with 70 m cell strainer filter to remove any remaining clumps of cells. Count viable cells after staining with 0.4 Trypan blue, using a hemocytometer. From a nearly confluent well, 1-2 x 106 single hES cells can be obtained.

Immunofluorescence Labeling Of Cultured Cells

Cells of interest, growing in tissue culture Trypsin EDTA solution (e.g., Life Technologies) 2 formaldehyde (see recipe) Phosphate-buffered saline (PBS see recipe), pH 7.4 PBS FBS PBS, pH 7.4, containing 10 fetal bovine serum (FBS) 0.1 (w v) saponin in PBS FBS prepare fresh from 10 (w v) saponin stock solution (store stock up to 2 months at 4 C or in aliquots up to 1 to 2 years at -20 C) Primary antibody

Targets for Radioactive Assays 327321 G proteincoupled receptor

The SPA technology can also be applied for kinase assays by immobilizing the substrate on the beads.15'21-24 Here, a biotinylated peptide substrate was captured by streptavidin-coated SPA beads. This design is not working for all kinases and alternative designs were developed as described for protein kinase A (PKA).16 In this study avidin-coated polylysine YSi beads were used for capturing a biotinylated PKA substrate peptide. The assay was optimized for the 384-well format with regard to the following parameters (1) amount of beads (2) avidin concentration and (3) ATP concentration. With the optimized conditions, the Km values for the substrate and ATP were determined and found to be close to the those reported in the literature. Enzyme titration as well as kinetic studies were performed, which showed that the reaction runs in a linear fashion for up to 100 min. Assay development was concluded by determining the effects of EDTA, DMSO, and the stability of the 'stopped reaction mix'...

Substrate specificity

Similar problems may arise during investigations of DPO inhibitors. There is much interest in these, since the food industry is keenly aware of the need to control enzymic browning in many foods. Inhibition of enzymic browning can be brought about (1) by reversing or blocking the later color-producing reactions or (2) by inhibiting the actual DPO enzyme (usually a catecholase). Examples of the former include the control of enzymic browning in fruit juices by addition of ascorbic acid, which reduces the quinoid reaction products back to the parent phenol (Embs and Markakis, 1965 Golan-Goldhirsh and Whitaker, 1984), or cysteine, which combines with quinones to form colorless thioethers, thus blocking any further reaction to form colored end-products (Walker and Reddish, 1964 Montgomery, 1983 Dudley and Hotchkiss, 1989 Richard et al., 1991). By contrast, true inhibition of a DPO will involve some sort of interactive effect with the active site of the enzyme, for example, inhibition by...

Other CAM Therapies

Multiple CAM therapies are unstudied or unlikely to be beneficial at the same time, they are possibly unsafe, expensive, or labor-intensive. Because of these concerns, these therapies should be fully investigated and well understood before use calcium EAP, Candida (yeast) therapy, chelation therapy, dental amalgam removal, DHEA, hyperbaric oxygen, Prokarin, and toxin avoidance.

Harvesting Attachmentdependent Cell Cultures From Tissue Culture Flasks

0.05 to 0.25 trypsin solution with 1 mM EDTA or cell dissociation solution (Invitrogen) 5. Add trypsin-EDTA solution to barely cover cells. Incubate at room temperature or 37 C until cells loosen. 7. Add equal volume of warm complete medium as the amount of trypsin-EDTA solution to each flask and pipet over the surface to remove and suspend cells.

Dot And Slot Blotting Of Dna Onto A Positively Charged Nylon Membrane Using A Manifold

Positively charged nylon membrane (see Table A.4G.1) 0.4 M and 1 M NaOH (appendix 2e) 200 mM EDTA, pH 8.2 (appendix 2e) 2x SSC (appendix 2e) 3. Add 1 M NaOH and 200 mM EDTA, pH 8.2, to each sample to give a final concentration of 0.4 M Na0H 10 mM EDTA. Denature 10 min at 100 C. Microcentrifuge each tube 5 sec.

Preparation Of Lox Extracts

Methods are given to homogenize either dried tissue or fresh tissue containing a high percentage of water. Centrifugation of the homogenate gives a solution containing LOX activity. Appropriate homogenizing buffers (50 mM, pH 6 to 8) include 2-(N-mor-pholino)ethanesulfonic acid (MES), acid) (PIPES), acid (HEPES), or TrisCl. One can also use 0.1 M sodium acetate, pH 4.5, with certain seeds (e.g., soybeans see Axelrod et al., 1981). Optional amendments with polyvinylpolypyrrolidone (PVPP) polyvinylpyrrolidone (PVP), phenylmethylsulfonyl fluoride (PMSF), sodium metabisulfite, ascorbic acid, Triton X-100 Brij 99, or EDTA may be desirable (see Critical Parameters).

Preparation Of Inactivated Sendai Virus Stock

Adjust infectious Sendai virus stock to 4.21 mg protein ml in prechilled 1 mM EDTA in PBS. Record the volume of this diluted stock. 8. Resuspend pellet in the original volume of 1 mM EDTA in PBS (step 1). Verify inactivation by plaque assay (see Support Protocol 4) and determine protein concentration (e.g., Pierce or Bio-Rad).

Crystallization of Native Proteins

Figure 1 Size-exclusion chromatography with an apocarotenoid-15,15'-oxygenase using a Superdex 200 column.27 (a) Run without detergent under normal protein preparation conditions (50 mM Tris-HCl at pH 8.0, 200 mM NaCI, 1 mM EDTA, 2mM DTT). (b) Run under the same conditions but with 0.25 C8E4-8 (octylpolyoxyethylene). In SDS-PAGE, all fractions below 100 mL contained exclusively the prepared enzyme with a polypeptide mass of 54 kDa. The run without detergent shows monomer (46 kDa), dimer (90 kDa), and aggregates (void). The run with detergent shows micelles (150 kDa) and aggregates (void). (Reproduced with permission from Science - Supplementary Material.) Figure 1 Size-exclusion chromatography with an apocarotenoid-15,15'-oxygenase using a Superdex 200 column.27 (a) Run without detergent under normal protein preparation conditions (50 mM Tris-HCl at pH 8.0, 200 mM NaCI, 1 mM EDTA, 2mM DTT). (b) Run under the same conditions but with 0.25 C8E4-8 (octylpolyoxyethylene). In SDS-PAGE, all...

Antidotes and treatment of poisonings

Chelating agents (A) serve as antidotes in poisoning with heavy metals. They act to complex and, thus, inactivate heavy metal ions. Chelates (from Greek chele claw of crayfish ) represent complexes between a metal ion and molecules that carry several binding sites for the metal ion. Because of their high affinity, chelating agents attract metal ions present in the organism. The chelates are non-toxic, are excreted predominantly via the kidney, maintain a tight organometallic bond also in the concentrated, usually acidic, milieu of tubular urine and thus promote the elimination of metal ions. Na2Ca-EDTA is used to treat lead poisoning. This antidote cannot penetrate cell membranes and must be given parenterally. Because of its high binding affinity, the lead ion displaces Ca2+ from its bond. The lead-containing chelate is eliminated renally. Nephrotoxicity predominates among the unwanted effects. Na3Ca-Pentetate is a complex of dieth-ylenetriaminopentaacetic acid (DPTA) and serves as...

Prepare the pancreatic samples

Centrifuge sample, rehomogenize (in the same volume as initially used of homog-enization medium containing DPPD), and recentrifuge, then pool supernatants (see Basic Protocol 1, steps 4 and 5). Add MES to 5 mM (from 1 M stock) and EDTA to 1 mM (from 0.2 M stock). Filter and disperse with a Dounce homogenizer (see Basic Protocol 1, step 5). Maintain homogenized sample (PNS) at 0 C, preferably briefly, until loaded on the gradients.

Gelatinase B

Collier et al59 expressed gelatinase B residues 93-708. IBs were solubilized in 8 M urea 25 mM Tris-HCl (pH 7.5) 5 mM CaCl2 1 mM PMSF 0.2 M NaCl 10 mM EDTA. Soluble protein was obtained after dialysis against buffers containing successively, 6, 4, 2 and 0 M urea. The active recombinant protein was purified by gelatin-agarose chromatography.

Cmffbsdte solution

If such agents are not desired (e.g., for certain biochemical or functional analyses), 0.1 mM EDTA can be substituted for DTE. CMF FBS EDTA solution 45 ml 10x HBSS 50 ml FBS 7.5 ml 1 M HEPES 10 ml 0.25 M EDTA H2O to 500 ml 100 g ml gentamycin Adjust pH to 7.2 with HCl Prepare fresh The 0.25 M (50x) EDTA stock solution can be made in advance and stored indefinitely at 4 C.

Maintain the culture

Wash cells once with PBS or trypsin EDTA to remove remaining serum from the cells by covering cells with the solution and pipetting it off. 6. Overlay cells with 37 C trypsin EDTA using a volume that is just enough to cover the monolayer (e.g., 0.3 ml for a 25-cm2 flask). Allow to sit 30 to 40 sec (cells should become detached) and shake the flask to completely detach cells.

Lysis of Bacteria

The following lysis protocol is a combination of freezing-thawing, lysozyme activity, and mechanical disruption by sonification and potter homogenization. All steps, except the lysozyme incubation, should be performed on ice. The relatively high EDTA concentration destabilizes membranes and inhibits metal proteases, whereas PMSF and pefabloc inhibit serine proteases. 3. While stirring, add the following components 100 L NP-40, 50 L PMSF solution, 200 L pefabloc solution, 10 mL EDTA solution, and 10 mL freshly prepared lysozyme solution.

Treatment Method

In chelation therapy, ethylenediaminetetraacetic acid (EDTA) is given through an intravenous infusion. EDTA binds strongly to (chelates) harmful metals, and the metal-EDTA complexes then are excreted in the urine. Vitamin and mineral supplements are also frequently given. A course of treatment may involve 20 to 30 infusions that are given over the course of a few months. This type of therapy is effective for known situations of heavy-metal toxicity, such as lead poisoning. Some chelation products can be taken orally. These are of no proven value, and the U.S. Food and Drug Administration (FDA) has determined that they should not be sold.

Extracting Protein

The extraction process should completely disaggregate and denature the protein into unfolded monomers. Urea is not recommended for the initial extraction. For example, even if it is known that a native version of protein can be unfolded with 4 M urea, the same protein in an E. coli inclusion body will almost certainly not be completely extracted as unfolded monomers with that same concentration of urea (or in most cases, even with 8 M urea). Initial extraction trials should be carried out with guanidineHCl, which is more effective than urea. Most proteins will be extracted with 6 to 8 M guanidineHCl. There should be adequate reductant present to maintain sulfhydryl groups in the reduced state, and thus prevent artificial disulfide bond formation. The presence of EDTA and a slightly acidic pH of 6.0 to 6.5 will help minimize cysteine oxidation. The extract may require clarification by filtration or centrifugation.

Antigen Retrieval

The following is called the Steam EDTA Protocol. It is a heat-induced 2. Place a reagent reservoir containing 200 mL of 1 mM EDTA, pH 8.0 in the upper chamber of the steamer. 4. After the distilled water in the lower chamber of the Black & Decker Handy Steamer has reached a full boil ( 10-12 min) place the slides into the 1 mM EDTA in the upper chamber of the steamer.

Causes of anemia

Thalassemia (microcytic, hypochromic) must be differentiated from iron deficiency. Iron levels are normal in thalassemia iron is contraindicated because it may cause overload. Look for elevated hemoglobin A, (p-thalassemia only) or hemoglobin F (J3-thalassemia only), target cells, nucleated RBCs, diffuse basophilia on peripheral smears, x-ray of the skull showing a crew-cut appearance, splenomegaly, and positive family history (more common in blacks, Mediterraneans, Asians). No treatment is required for minor thalassemia patients often are asymptomatic as they are used to living at a lower level of hemoglobin and hematocrit. Thalassemia major is more dramatic and severe. Treat with as-needed transfusions and iron chelation therapy to prevent hemochromatosis. Diagnosis is made by hemoglobin electrophoresis. There are four gene loci for alpha-chain and only two for beta -chain thalassemia. Alpha thalassemia is symptomatic at birth, or the fetus dies in utero (hydrops) beta thalassemia...