Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Polyacrylamides”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,099 records · Page 61Linked to original sources
PubMed1990

Determination of poly(ADP-ribose) chain length distribution on polyacrylamide gels by silver staining.

Silver staining and polyacrylamide gel electrophoresis were used to visualize chain length distribution of poly(ADP-ribose) enzymatically synthesized from NAD by rat liver nuclei. The method described has the advantage that synthesis does not require radioactive-labeled NAD, and microgram quantities (greater than 5 micrograms) of poly(ADP-ribose) can be resolved and visualized as discrete bands according to chain lengths which range from 8 to 60 residues. This method can be applied to estimate size distribution of poly(ADP-ribose) chains in cells or tissues.

Chromatography↗
PubMed1990

Enhanced removal of detergent and recovery of enzymatic activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis: use of casein in gel wash buffer.

The inclusion of 1% casein or bovine serum albumin in buffer used to reactivate enzymes subjected to sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis resulted in accelerated removal of SDS and restoration of nuclease and beta-galactosidase enzyme activities. Nuclease and beta-galactosidase activities which are absent from gels after longer wash procedures are detectable with this technique. Enzyme activity in gels prepared with SDS which contained inhibitory contaminants was partially restored by the casein wash procedure. The threshold of detection of two-dimensionally separated deoxyribonuclease I using the casein wash procedure was 1 picogram.

Autoradiography↗
PubMed1990

Electrotransfer of basic proteins from nondenaturing polyacrylamide acid gels to nitrocellulose: detection of enzymatic and inhibitory activities and retention of protein antigenicity.

We have developed a method for electrotransfer of strongly basic proteins (lysozyme, pI 11; mucus proteinase inhibitor, pI greater than 10; bovine pancreas trypsin inhibitor; pI 10.5; human leukocyte elastase, pI greater than 9) from nondenaturing acid gels (pH 4.5) to nitrocellulose sheets. Buffers were those used in a discontinuous system for transfer from sodium dodecyl sulfate (SDS)-containing polyacrylamide gels with one modification in the cathode buffer which contained 0.1% SDS. This method was compared to electrotransfer performed in 0.7% acetic acid. The basic proteins studied, which were positively charged in the gel, formed with SDS negative complexes which migrated toward the anode and were efficiently transferred to the nitrocellulose. Moreover, their biological properties were preserved: inhibitory activity, enzyme activity, and antigenicity. This method is advantageous because it is simple, is sensitive, and can be applied to various biological fluids to detect inhibitors, enzymes, and other proteins which have a basic character, after electrophoretic separation under their native forms.

Animals↗
PubMed1990

Dispersal of proteolipid macroaggregates with trifluoroacetic acid and analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

The propensity of highly purified proteolipids to form macroaggregates in aqueous solutions, especially when heated with sodium dodecyl sulfate (SDS), with or without thiol reagents, has made qualitative and quantitative analyses of individual species by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) difficult and unreliable. Comparisons of proteolipid profiles from liver, brain, and cultured human keratinocytes demonstrate that 40-72% of the total proteolipid in SDS-PAGE sample buffer is in the form of macroaggregates. Treatment of proteolipids with neat trifluoroacetic acid (TFA) followed by removal of the TFA and incubation in cold SDS-PAGE sample buffer causes complete dispersal of the macroaggregates and allows recovery of virtually all of the proteolipid applied to gels (increasing yields by as much as 3.6 times, depending on tissue type). Gels of TFA-treated samples display differences not only in the relative amounts of individual species but also in novel species not found in untreated samples. Eluted macroaggregates treated with TFA display the same SDS-PAGE banding profiles as TFA-treated whole proteolipids. Hence, routine TFA treatment of proteolipids prior to SDS-PAGE increases total proteolipid yields, allows reliable quantitation of individual apoprotein species, and reveals species previously obscured by the formation of macroaggregates.

Animals↗
PubMed1990

Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes.

A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as [35S]methionine-labeled proenkephalin or 125I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of [35S]proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved [35S]methionine-labeled proenkephalin but not 125I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.

Adrenal Medulla↗
PubMed1990

Direct carbohydrate analysis of glycoproteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate-polyacrylamide gel.

A procedure for the carbohydrate analysis of glycoproteins electrotransferred to a polyvinylidene difluoride membrane is described. The glycoproteins (plant lectins, transferrin, and vitronectin) were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a membrane. Each of the glycoprotein bands visualized by staining with Coomassie brilliant blue R-250 was excised from the membrane and subjected to direct hydrolysis either in 2.5 M trifluoroacetic acid at 100 degrees C for 6 h for neutral sugars and hexosamines, or in 0.05 M H2SO4 at 80 degrees C for 1 h for sialic acids. The hydrolysate obtained was analyzed for neutral sugars, hexosamines, and sialic acids independently by three different systems of high-performance liquid chromatography. The analytical values were reproducible with reasonable accuracy and agreed with those expected with recoveries of 57-66%. The method was successfully applied to a mannose-specific lectin of Sophora japonica bark, which is composed of four different subunits that aggregate sugar specifically. Because the four subunits could be separated by SDS-PAGE alone, the method proved useful for determining their carbohydrate compositions. Three of them were shown to contain carbohydrates typical of N-linked oligosaccharides of plant origin, which agreed well with the results of the binding assay carried out on a membrane using various horseradish peroxidase-labeled lectins.

Carbohydrates↗
PubMed1990

Modification of a discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide gel system for minigel electrophoresis.

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system was recently described by J. P. Doucet and J. M. Trifaró [1988) Anal. Biochem. 168, 265-271). The system was developed to separate with high and broad resolution the components from large volume samples after an overnight electrophoresis. This system was found to have many advantages. However, when used directly as a minigel system, this method cannot sustain the high voltage inherent to minigel electrophoresis and produces artefacts, namely a double front and a loss of resolution in the low molecular weight range. These problems were eliminated using the buffer system of M. A. Porzio and A.M. Pearson [1977) Biochem. Biophys. Acta 490, 27-34) in the separating gel and in the electrode chambers. The resulting modified discontinuous minigel system has the same advantages as the technique described for large slab gel electrophoresis, including the effective and rapid transfer of high molecular weight proteins to nitrocellulose membranes, as well as the advantages of the minigel format.

Animals↗
PubMed1990

Production and separation of peptides from proteins stained with Coomassie brilliant blue R-250 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Proteins stained with Coomassie brilliant blue on polyacrylamide gels were digested with lysylendopeptidase in the presence of sodium dodecyl sulfate. Peptide production was similar to that under ordinary conditions of digestion. Peptides were recovered easily and efficiently from the gel pieces and separated by HPLC. The present method for preparation of peptides from proteins separated by sodium dodecyl sulfate gel electrophoresis is quite simple and can be used for sequence analysis of proteins in general at the subnanomolar level.

Amino Acid Sequence↗
PubMed1990

Silver staining of extensively glycosylated proteins on sodium dodecyl sulfate-polyacrylamide gels: enhancement by carbohydrate-binding dyes.

Two methods are described for detecting less than 1 microgram of highly glycosylated proteins, such as mucins, on sodium dodecyl sulfate-polyacrylamide gels. They combine commonly employed periodic acid-Schiff (PAS) and Alcian blue dyes with silver stain. Carbohydrate prestaining renders mucins more cationic and favors greater complexation with ionic silver. Comparisons of different mucin samples stained either with PAS-silver or alcian blue-silver indicate differential staining between the two techniques. Such differences may, in part, be due to an affinity of Alcian blue for sulfated glycoproteins. These two staining protocols when used in conjunction with silver staining alone are particularly valuable for assessing sample purity and for detecting contaminating proteins during mucin purification protocols.

Alcian Blue↗
PubMed1990

Affinity purification of polyclonal antibodies from antigen immobilized in situ in sodium dodecyl sulfate-polyacrylamide gels.

A procedure for the preparation of affinity-purified antibody is described. Protein mixtures are separated by electrophoresis in sodium dodecyl sulfate (SDS)--polyacrylamide gels. Individual bands of protein are cut from the gel and fixed in situ with glutaraldehyde. The gel pieces are then homogenized and washed extensively with buffered solutions and chaotropic agents. The washed gels can then be used as immunoadsorbents to purify antibodies from crude antisera. This method should be especially useful for the preparation of small amounts of antibody to proteins that are difficult to purify by conventional means, that are available only in limited quantity, or that cannot be blotted to immunoadsorbents such as nitrocellulose or diazotized paper.

Animals↗
PubMed1990

Native agarose-polyacrylamide gel electrophoresis allowing the detection of aminopeptidase, dehydrogenase, and esterase activities at the nanogram level: enzymatic patterns in some Frankia strains.

Nanogram amounts of soluble aminopeptidases, dehydrogenases, and esterases were detected by nondenaturing ultralow gelling point agarose-polyacrylamide gel electrophoresis (ULGA-PAGE). Cytosolic fractions from Frankia sp. were electrophoresed at 4 degrees C in the presence of Co2+, Zn2+, or Mg2+ ions. Then, aminopeptidases and esterases were revealed by simultaneous capture staining by using fast garnet GBC diazonium salt as the chromogenic coupling compound. Dehydrogenases were revealed by using nitro blue tetrazolium salt as electron acceptor. A variety of aminopeptidases, dehydrogenases, and esterases could be identified by their migration in ULGA-PAGE and by their sensitivities to NaCl, CoSO4, ZnSO4, and MgCl2 when assayed "ingel." The presence of agarose was essential for the detection of the complex enzyme patterns. The patterns were remarkably similar for the five Frankia strains isolated from Allocasuarina and Casuarina host plants and differed from those of Frankia strains isolated from Comptonia and Hippophaë host plants. A nomenclature is proposed for aminopeptidases and other Frankia enzymes. The richness of the Frankia amino-peptidases and esterases zymograms makes them adequate marker enzymes for taxonomical, genetic, or biochemical studies. Dehydrogenases might also be useful, although a more restricted number of bands were found with L-lactic and L-malic acid as substrates.

Actinomyces↗
PubMed1990

Determination of sulfhydryl groups and disulfide bonds in a protein by polyacrylamide gel electrophoresis.

A general method by polyacrylamide gel electrophoresis for the determination of sulfhydryls and disulfides in a protein was developed. The method included a two-step alkylation procedure: the first step consisted of alkylation of the sulfhydryl groups with iodoacetic acid in the presence and absence of 8 M urea; the second step consisted of alkylation of the disulfide groups with iodoacetamide after reduction with a thiol. By high-pH urea gel electrophoresis, all the half-cystine residues in a protein could be categorized into three states: reactive sulfhydryls, nonreactive sulfhydryls, and disulfide bonded. The particular advantage of the method is that the states of half-cystines in different protein species can be analyzed independently both in isolated protein and in biological translation systems.

Alkylation↗
PubMed1991

Detecting proteins containing 3,4-dihydroxyphenylalanine by silver staining of polyacrylamide gels.

Proteins in which some or all of the tyrosine side chains are post-translationally modified to dihydroxyphenylalanine have been found in several invertebrate phyla. In this paper we describe the unusual silver-staining properties of these 3,4-dihydroxyphenylalanine (Dopa)-proteins in silver-stained polyacrylamide gels. Our evidence suggests that the rapid silver staining of these proteins is due to the 3,4-dihydroxyphenol ring which is a highly effective reducing agent in the alkaline development conditions used in the final step of most silver-staining procedures. Normal proteins comprising the standard 20 amino acids and tyrosine on its own, do not reduce silver under these conditions. Pretreatment of the gels with acid-dichromate solutions abrogates the rapid staining of the Dopa-proteins. This rapid silver-staining technique will facilitate the rapid screening of many additional organisms for Dopa-proteins using sodium dodecyl sulfate gels and small amounts of tissue.

Animals↗
PubMed1991

Direct tissue isoelectric focusing on mini ultrathin polyacrylamide gels followed by subsequent western blotting, enzyme detection, and lectin labeling as a tool for enzyme characterization in histochemistry.

Application of cryostat sections directly onto mini ultrathin polyacrylamide isoelectric focusing gels allows an elution of proteins out of the sections into the gels under conventional focusing conditions. Protein bands representing alkaline phosphatases can easily be identified on nitrocellulose after performing a modified Western blot procedure. Furthermore, carbohydrate residues of several isoforms of alkaline phosphatases separated by isoelectric focusing can be demonstrated in a single blot strip by subsequent incubation of this strip with substrates for alkaline phosphatase and peroxidase, the latter being employed as the enzyme to which the applied lectins are covalently linked. This simple and reproducible procedure is likely to enable histochemists to determine isoforms of enzymes from a single cryostat section.

Alkaline Phosphatase↗
PubMed1991

A method for quantifying radioactivity associated with protein in silver-stained polyacrylamide gels.

A method is described in which individual proteins labeled with weak beta-emitting radionuclides, separated by polyacrylamide gel electrophoresis, and stained with silver are released from the gel by the use of the periodate soluble cross-linking agent N,N'-dialyltartardiamide. The radioactivity can then be quantitated using liquid scintillation counting. The method is shown to be insensitive to reasonable variations in the intensity of staining as well as the gel volume over a practical range of gel slices. Recovery from the gel is extremely good with 93% of the counts associated with 14C-labeled proteins of known radioactive concentration being recovered. Analysis of a complex mixture of 3H-labeled proteins indicates resolution similar to that obtainable by autoradiography without the problems associated with quenching of autoradiographic signal by the staining procedure. The method is used to determine the amount of fucose and mannose incorporated into a putative cell adhesion protein during development of the cellular slime mold Dictyostelium purpureum.

Autoradiography↗
PubMed1991

Single-step electroelution of proteins from SDS-polyacrylamide gels and immobilization on diisothiocyanate-glass beads in prepacked capillary columns for solid-phase microsequencing.

A new method for immobilization of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to sequencing is described. It utilizes a simple apparatus that permits the simultaneous electroelution of proteins from gel slices and attachment to diisothiocyanate-activated glass beads prepacked in capillary tubes [S-P. Liang and R. A. Laursen, Anal. Biochem. 188, 366-373 (1990)]. Transfer/attachment yields of greater than 80% within 90 min were observed for several 125I-labeled proteins with a range of molecular weights using 0.2 M sodium phosphate (pH 8.9) buffer containing 0.1% SDS. The method has the advantage of high capacity, relative simplicity, and insensitivity to the presence of SDS and Coomassie blue stain. The highest transfer yields were obtained when proteins were run on gels which had been aged for at least 12 h. For 100- to 1000-pmol samples, the sequenceable amount of protein, including transfer, was generally 30-60%, with an average repetitive yield of 95%. Factors which influence sample recovery and sequencing yield are discussed.

Amino Acid Sequence↗
PubMed1991

Structural analyses of proteins electroblotted from native polyacrylamide gels onto polyvinyldiene difluoride membranes. A method for determining the stoichiometry of protein-protein interaction in solution.

The usefulness of a native gel electroblotting technique in the study of protein-protein interactions was demonstrated by the determination of the stoichiometry of the interaction between interleukin-2 (IL-2) and the alpha subunit of IL-2 receptor (IL-2R alpha) in solution. Complexes formed between the recombinant forms of the two proteins in solution were separated from the noncomplexed protein molecules by electrophoresis in a native polyacrylamide gel and the protein bands were electroblotted quantitatively onto polyvinyldiene difluoride membranes for further structural analysis. The data obtained from sequence and amino acid analyses of the blotted proteins provided direct evidence that IL-2 binds to IL-2R alpha in a 1:1 ratio. This methodology should be applicable to the study of other structure/function aspects of protein-protein interactions in solution.

Amino Acid Sequence↗
PubMed1992

A specific stain for the detection of nonheme iron proteins in polyacrylamide gels.

Nonheme iron proteins can be visualized as blue bands in native polyacrylamide gels using a staining method that is both simple and rapid. The reaction of potassium ferricyanide with protein-bound iron atoms to form royal blue complexes occurs almost instantaneously and is sensitive enough to detect 1 microgram of analytical-grade ferritin and 2 micrograms of purified ferredoxin from cyanobacteria. No special treatment of reagents or apparatus was necessary. On comparison, this stain was found to be more specific than the Ferene S stain, not detecting bovine serum albumin even when present as a hundredfold excess over ferritin. The method was found to be effective for isoelectric focusing gels as well.

Animals↗