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At least 145 records · Page 8Linked to original sources
PubMed1994

Polyacrylamide gradient gel electrophoresis as a method to measure transfers of radiolabeled cholesteryl esters between several plasma lipoprotein fractions.

In human plasma, cholesteryl esters can redistribute between various lipoprotein fractions through an exchange reaction mediated by the cholesteryl ester transfer protein. In the present study, liquid scintillation counting of polyacrylamide gradient gel fragments was applied to the determination of transfers of radio-labeled cholesteryl esters between different lipoprotein fractions. This method consisted in four successive steps: (1) incubation of total human plasmas with a tracer dose of high-density lipoproteins (HDL) containing tritium 3H-labeled cholesteryl esters, (2) separation of plasma lipoprotein fractions by electrophoresis in 20-160 g/liter polyacrylamide gels, (3) cutting off and dissolution with a NaOCl solution of polyacrylamide gel fragments which contained the different lipoprotein fractions, (4) liquid scintillation counting of the dissolved gel fragments. The present method provided a convenient and accurate method of determining the rate of cholesteryl ester transferred between several plasma lipoprotein classes. As an example, it was applied to the measurement of the rates of radiolabeled cholesteryl esters transferred from either HDL3 or HDL2 toward the two other main plasma lipoprotein fractions, very-low-density lipoproteins and low-density lipoproteins.

Cholesterol Esters↗
PubMed1995

The use of preparative polyacrylamide gel electrophoresis and electroelution for purification of mucus glycoproteins.

This paper describes a novel technique for purifying glycoproteins from porcine gastric mucus by preparative polyacrylamide gel electrophoresis and electroelution. The method is based on the observation that the high-molecular-weight buffer/SDS-soluble mucins do not penetrate through the polyacrylamide gel, but remain on the gel surface. Mucus solution extracted with 6 M urea was fractionated on Sepharose CL-2B column and Vo peak mucin was submitted to purification by preparative polyacrylamide gel electrophoresis (22 h). Nonpenetrated mucin layer was electroeluted from the gel after the reversing of electrode polarity (3 h). A comparison of mucin preparations purified by our method and by CsCl density gradient centrifugation indicated that the GalNAc/protein and GalNAc/DNA ratios were three times higher than those of the first method. The method is a relatively short and efficient procedure and yields pure mucin preparation free of contaminating proteins and nucleic acids.

Acetylgalactosamine↗
PubMed1982

A polyacrylamide-gel electrophoretic study of human tear proteins.

The protein composition of normal and pathological tears was studied by polyacrylamide-gel (disc) electrophoresis. Polyacrylamide-gel electrophoresis was shown to detect at least 14 fractions in 2-10 microliters of native tears. A comparison was made between the protein composition of normal tears and serum. The most characteristic bands in the tear-protein pattern were identified by parallel electrophoresis of tears, serum, human milk and purified egg-white lysozyme. The identified fractions were specific tear prealbumin, serum albumin, transferrin, lactoferrin and lysozyme. An unidentified major tear-protein component was also described. The tear-protein pattern was divided into six zones: (1) prealbumin zone; (2) post-albumin zone; (3) post-transferrin zone; (4) macroglobulin zone; (5) basic globulin zone; (6) prelactoferrin zone. A significant rise in the level of serum albumin and transferrin was demonstrated in tears from cases of acute catarrhal conjunctivitis. The optimal circumstances were discussed under which major and minor tear components and basic and acidic tear proteins can be determined simultaneously. Polyacrylamide-gel electrophoresis is recommended as a useful method to study the various diseases of the anterior segment of the eye.

Electrophoresis, Polyacrylamide Gel↗
PubMed1978

A simplified polyacrylamide gel electrophoresis apparatus for simultaneous application of multiple buffer systems or detergent combinations.

A previous design of an apparatus for the simultaneous fractionation by polyacrylamide gel electrophoresis in 10 different buffer systems (1) was replaced by a greatly simplified new design, employing small, cylindrical buffer partitions within the lower buffer reservoir and/or upper buffer reservoir of a conventional, temperature-regulated polyacrylamide gel electrophoresis apparatus for cylindrical gels. The apparatus was tested in application to the problem of simultaneous polyacrylamide gel electrophoresis in different buffer systems with the purpose of optimizing the operative pH for a particular fractionation problem. It was also applied to fractionations in a single buffer system to which various combinations of ionic and nonionic detergents were admixed.

Adenylyl Cyclases↗
PubMed1983

Multiple forms of amylase in leaf extracts: electrophoretic transfer of the enzyme forms into amylose-containing polyacrylamide gels.

A method for the analysis of multiple forms of glucan-degrading enzymes is described. The procedure consists of the separation of the proteins by electrophoresis or isoelectric focusing in glucan-free polyacrylamide gels followed by the nondenaturing electrophoretic transfer into a second polyacrylamide layer which contains immobilized glucans. The method combines the resolving power of electrophoretic separations in glucan-free media with the sensitivity of amylase activity detection in amylose-containing polyacrylamide gels. The procedure is especially useful when samples containing low amylase activity, but a large number of multiple enzyme forms, are to be analyzed.

Amylases↗
PubMed1983

High sensitivity method for fluorofore detection in gradient polyacrylamide slab gels through excitation by laser light: application to glycoproteins stained with concanavalin A-fluorescein isothiocyanate.

An easy-to-assemble apparatus for the laser-light excitation of fluorofores in polyacrylamide gels is described. The assemblage is made up of a continuous-wave ion-argon laser with adjustable power output, a beam diffuser, appropriate filters to block excitation light, and a photographic camera. With this setup a minimum 20-fold increase of sensitivity was obtained for fluorofore detection in polyacrylamide gels as compared to the more conventional uv-light excitation using a commercial preparation of Con A-FITC (concanavalin A-fluorescein isothiocyanate) as reference molecule in the gel. The same apparatus, used to analyze the Con A-positive glycoproteins contained in serum Cohn fraction IV separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed a number of fluorescent components in a wide range of relative intensities while uv-light excitation showed none. Acrylamide concentration in the gel is critical, since a working limit of between 10 and 12% has been found, above which the diffusion of Con A-FITC in the gel, necessary to label glycoprotein bands, is hampered. The system described here also permits the optimization of detection of minor components not otherwise observable by conventional light excitation, because light power, angle of incidence, and beam divergence can be adapted to analyze specific areas of the sample gel.

Concanavalin A↗
PubMed1985

Peptide mapping of basic proteins by proteolysis in acetic acid/urea-minislab polyacrylamide gels.

A method to obtain peptide maps of basic proteins on acetic acid/urea (AU) -polyacrylamide minislab gels is presented. Basic proteins such as the histones are digested with Staphylococcus aureus V8 protease in the stacking gel (pH 4) of an AU-polyacrylamide minislab gel. As the peptides are resolved in the AU minislab gel on the basis of charge and size, it is possible to separate peptides containing modified amino acids from the unmodified, parent peptide. The peptide(s) containing the modified residue may be identified following electrophoresis on a second-dimension sodium dodecyl sulfate-polyacrylamide minislab gel. This procedure will be useful for comparing histone variants and for the study of histone modifications.

Acetates↗
PubMed1985

Purification and characterization of keratin hydrolase in psoriatic epidermis: application of keratin-agarose plate and keratin-polyacrylamide enzymography methods.

Keratin-agarose plate and keratin-polyacrylamide enzymography methods were developed to demonstrate proteolytic digestion of epidermal keratin. By applying these methods, keratin hydrolase was purified from Tris-buffered saline extract of psoriatic scales by 50% ammonium sulfate precipitation, passage through a lysine-Sepharose column, DEAE-Sepharose, Sephacryl S-200, high-performance cation-exchange chromatography on Mono S, and aprotinin-Sepharose affinity chromatography. The final preparation demonstrated a single protein band at molecular weight 30,000 judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, in keratin-polyacrylamide slab gels, the purified enzyme preparation showed a translucent band at molecular weight 30,000, indicating keratin digestion. Keratin hydrolase digested reassembled epidermal keratin as well, whereas it had no effect on guinea pig hair keratin. The enzyme demonstrated a high level of hydrolytic activity on Ile-Pro-Arg-p-nitroanilide and other peptidyl arginine substrates, while it showed a low level of activity on Val-Leu-Lys-p-nitroanilide, and no activity on Arg-Pro-Tyr-p-nitroanilide, Glu-Pro-Val-p-nitroanilide, or Ala-Ala-Ala-p-nitroanilide. The keratin hydrolase was a serine proteinase, inactivated by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, tosyl-lysyl-chloromethyl ketone, antipain, leupeptin, soybean trypsin inhibitor, aprotinin, and p-aminobenzamidine. The keratinolytic activity was not detected in normal epidermal extract.

Adult↗
PubMed1985

A method to quantitate Coomassie blue-stained proteins in cylindrical polyacrylamide gels.

A method for the quantitation of Coomassie blue-stained proteins in cylindrical polyacrylamide gels is described. It involves an elution of the dye with an 80% methanol solution in a sealed Pyrex tube at 100 degrees C for 3 h and a measurement of its concentration at 585 nm. Using a 6.5% polyacrylamide gel and bovine serum albumin as a protein standard, the curve of absorbance of the dye solution as a function of the amount of protein was observed to be linear up to 30-40 micrograms of protein and as little as 0.8-1.0 micrograms of protein could be measured. The validity of the method was indicated by the values obtained for the relative proportions of the human erythrocyte membrane proteins. Using this method, the color yields of several proteins varying widely with respect to their size, amino acid composition, and carbohydrate content were determined in a 6.5% polyacrylamide gel. The results showed that they were generally the same except for proteins having a high carbohydrate content which were significantly lower.

Animals↗
PubMed1986

Binding of lithium dodecyl sulfate to polyacrylamide gel at 4 degrees C perturbs electrophoresis of proteins.

Although polyacrylamide gel has no affinity to lithium dodecyl sulfate (LDS) at 25 degrees C, the gel maximally binds 17 mg of LDS per gram dry weight at 4 degrees C. When polyacrylamide gel electrophoresis is carried out at 4 degrees C in the presence of LDS instead of sodium dodecyl sulfate (SDS) using a continuous buffer system, migration of proteins with lower molecular weight is accelerated as a result of the deficiency of LDS in the frontal region of the gel. When the gel is saturated with LDS, electrophoresis in the presence of LDS at 4 degrees C shows a resolution higher than that of SDS-polyacrylamide gel electrophoresis at 25 degrees C.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Quantitative electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels onto positively charged nylon membranes.

A method for the reproducible and quantitative electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a single sheet of either Zetabind or Gene Screen Plus membranes is presented. This procedure uses commercially available equipment and includes three crucial parameters: the omission of methanol from the transfer buffer, the use of thin (0.75-mm) resolving gels, and a newly developed protocol for pretreatment of the polyacrylamide gel after electrophoresis and before electroblotting. This combination of parameters yields a blot that both qualitatively and quantitatively reflects the proteins in the original polyacrylamide gel.

Electrochemistry↗
PubMed1987

Polyacrylamide gel electrophoresis: reaction of acrylamide at alkaline pH with buffer components and proteins.

The chemical reaction of monomeric acrylamide with primary, secondary, and tertiary amines, used as buffer components in polyacrylamide gel electrophoresis systems, was investigated in the basic pH range. Adduct formation proceeded for several minutes up to weeks, depending on the reactivity of the amino groups. A pH shift in the reaction mixture due to an altered pK value of the reaction product was observed. However, a few primary amines (tris(hydroxymethyl)aminomethane, 2-amino-2-methyl-1,3-propanediol) and secondary amines 3-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid, 3-(dimethyl(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid) showed negligible shifts of pH. They are, therefore, useful as components in the polymerization mixture; whereas some tertiary amines showing complete pH stability as well (e.g., triethanolamine) are not suitable, as they acted as accelerators of gel polymerization. Acrylamide can also covalently bind to proteins by reacting with the epsilon-amino group of lysine residues, especially. Bovine serum albumin, having an acidic isoelectric point, and the basic protein cytochrome c were treated with different acrylamide concentrations at alkaline pH yielding modified protein molecules with altered electrophoretic mobilities in different polyacrylamide gel electrophoresis systems. This reaction gave rise to artifacts in alkaline polyacrylamide gels and isoelectric focusing systems when residual acrylamide monomers were still present in the gel matrix after the polymerization process ceased.

Amines↗
PubMed1988

Discontinuous polyacrylamide gradient agarose gels resolve a wide range of restriction fragments and optimize the efficiency of nucleic acid transfer.

A vertical electrophoresis procedure utilizing a discontinuous polyacrylamide gradient agarose gel was developed to resolve DNA fragments ranging in size from over 50 kb to less than 300 bp in length. The gel consisted of a polyacrylamide plug at the base of the gel followed by a gradient of agarose ranging from 0.3 to 0.9%. Restriction fragments migrated shorter distances than in a comparable polyacrylamide-0.3% agarose gel, and small fragments were retained. Southern transfer of DNA fragments from the gradient gel onto nitrocellulose was more efficient than transfer of fragments using a nongradient gel.

Blotting, Northern↗
PubMed1988

Dodecyl maltoside-sodium dodecyl sulfate two-dimensional polyacrylamide gel electrophoresis of chloroplast thylakoid membrane proteins.

A two-dimensional electrophoretic system has been developed for the separation of chloroplast thylakoid membrane proteins. This system incorporates nondenaturing polyacrylamide gel electrophoresis in the presence of the nonionic detergent dodecyl-beta-D-maltoside in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Thylakoid membranes isolated from Spinacia oleracea were solubilized in 1.0% dodecyl-beta-D-maltoside and separated in 4-7% linear acrylamide gradient tube gels which contained 0.05% dodecyl-beta-D-maltoside. After electrophoresis, the tube gels were equilibrated with a sodium dodecyl sulfate-containing equilibration buffer and applied to a 12.5-20% acrylamide linear gradient gel. The Lammelli buffer system was used in both dimensions. The two-dimensional gels were analyzed by staining sequentially with 3,3',5,5'-tetramethylbenzidine-H2O2, Coomassie blue, and silver staining. A number of protein components were identified on "Western blots" of these two-dimensional gels by immunological localization. Membrane protein complexes such as the light-harvesting chlorophyll a/b protein complex, photosystem I, photosystem II, the cytochrome b6/f complex and ribulose bisphosphate carboxylase appear to migrate as essentially intact complexes in the first dimension and appear as vertical series of resolved subunits in the second dimension. This technique complements isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis in providing additional information concerning the subunit composition of membrane protein complexes and may prove to be of general utility for studying the protein composition of other membrane systems.

Chloroplasts↗
PubMed1989

Detection of chitinase activity after polyacrylamide gel electrophoresis.

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.

Chitinases↗
PubMed1990

Detection of chitin deacetylase activity after polyacrylamide gel electrophoresis.

Mucor racemosus and Rhizopus nigricans were used as sources of chitin deacetylases. Crude protein extracts were subjected to polyacrylamide gel electrophoresis at pH 8.9 (Davis system) or 4.3 (Reisfeld system) under native conditions. After electrophoresis, an overlay gel containing 0.1% (w/v) glycol chitin as substrate was incubated in contact with the separation gel. Chitin deacetylase activity was revealed by uv illumination with a transilluminator after staining for 5 min in 0.01% (w/v) Calcofluor white M2R. Chitosan (deacetylated chitin) generated by chitin deacetylases appeared more fluorescent than the intact chitin embedded in the overlay gel. Chitosan in a separate overlay gel was also subjected to a nitrous acid treatment which specifically depolymerizes chitosan while leaving chitin intact. Hydrolysis of chitosan by nitrous acid followed by Calcofluor staining yielded dark (nonfluorescent) bands (chitin deacetylase activities) in the fluorescent chitin-containing gel. Both assays revealed the presence of several chitin deacetylases from Zygomycetes. The same assays were performed after denaturing electrophoresis in 12% (w/v) polyacrylamide gels containing 0.1% (w/v) glycol chitin. Enzymes were renatured in buffered 1% (v/v) purified Triton X-100. Chitin deacetylases with estimated molecular weights between 26,000 and 64,000 were detected after Calcofluor staining. The assays were also performed in two-dimensional gel electrophoretic systems. Chitin deacetylases can be rapidly revealed by using the assay involving the nitrous acid treatment. However, both assays (with and without nitrous acid treatment) should be run to conclusively demonstrate chitin deacetylase activity after polyacrylamide gel electrophoresis.

Amidohydrolases↗
PubMed1990

A high-yield method for the isolation of hydrophobic proteins and peptides from polyacrylamide gels for protein sequencing.

A methodological approach is described which allows the isolation of hydrophobic and hydrophilic proteins and peptides in high yield. The technique consists of (1) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) protein elution from polyacrylamide gels with an organic solvent mixture composed of formic acid/acetonitrile/isopropanol/H2O (50/25/15/10, v/v/v/v), and (3) purification of eluted proteins by size exclusion chromatography on a Superose 12 column using this organic solvent mixture as eluant. The efficiency of this technique was tested with radioactively labeled polypeptides. These proteins were reaction center from Chloroflexus aurantiacus, bacteriorhodopsin, halorhodopsin from Halobacterium halobium, bovine serum albumin, ovalbumin, alpha-chymotrypsinogen A, and cytochrome c. The elution recoveries from polyacrylamide gels were 77-95%; the final yield after chromatographic purification was still 67-76% (with one exception). Subsequent amino acid sequencing was possible without further sample treatment. The sensitivity of the method described was found to be at least 20-30 micrograms protein.

1-Propanol↗
PubMed1991

Electrophoretic separation of large proteoglycans in large-pore polyacrylamide gradient gels (1.32-10.0% T) and a one-step procedure for simultaneous staining of proteins and proteoglycans.

A procedure is described for the preparation of 1.32-10% polyacrylamide gradient gels. Loose polyacrylamide gel on the top side of the gradient was stabilized with a layer of 0.4% agarose gel which also formed sample wells. The upper limit of separation achieved in these gels was estimated to be approximately 2 X 10(6) using globular protein standards. However, large aggregating proteoglycans from cartilage which have a molecular weight range of 1-4 X 10(6) penetrate and separate in these gels. A simple one-step procedure is also described for simultaneous staining of proteins and large proteoglycans in polyacrylamide gels.

Animals↗