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PubMed1991

Fast and sensitive silver staining of DNA in polyacrylamide gels.

The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. (1981, Science 211, 1437-1438) was modified to reduce unspecific background staining and increase sensitivity (down to 1 pg/mm2 band cross-section). Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage phi X174 were maintained despite eliminating oxidation pretreatment of fixed gels and reducing silver nitrate concentration. Preexposure to formaldehyde during silver impregnation enhanced sensitivity and the inclusion of the silver-complexing agent sodium thiosulphate in the image developer decreased background staining. Higher formaldehyde concentration during image development resulted in darker bands with good contrast. The procedure almost halves the number of steps, solutions and experimental time required and can be used for the staining of DNA fragments in polyacrylamide gels bound to a polyester backing film by controlling temperature during image development. We have applied this improved staining procedure for the routine analysis of complex DNA profiles generated by DNA amplification fingerprinting (DAF).

Bacteriophages↗
PubMed1991

Detection of possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by protein blotting to damaged DNA-fixed membranes.

A novel method for detecting possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by blotting them onto a damaged DNA-fixed membrane is presented. To prepare the membrane, highly polymerized calf thymus DNA immobilized on a nylon membrane is damaged chemically. Enzymes, either homogeneous or crude, that are possibly involved in the priming step of DNA repair are fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and are renatured to active form by incubating the gel in an appropriate buffer. The renatured enzyme is then blotted onto the damaged DNA-fixed membrane, a process during which incision and/or excision are introduced to the damaged DNA by the enzymes. The incision and/or excision provide priming sites for repair DNA synthesis in the subsequent step in which the membrane is incubated with DNA polymerase in the presence of alpha-32P-labeled substrate. The site of substrate incorporation on the membrane reflecting the molecular weight of the repair enzyme is finally visualized by autoradiography. The present technique is established using Escherichia coli exonuclease III and a DNA-fixed membrane treated with bleomycin or acid-depurinated. By application of this method, a priming factor (an exonuclease) involved in the initiation of bleomycin-induced DNA repair is detected in the extract of mouse ascites sarcoma cells, and thus the molecular weight of the enzyme is estimated. Some apurinic/apyrimidinic endonucleases of mammals are also detected by the present procedure.

Animals↗
PubMed1991

Detection of (2'-5')oligoadenylate binding proteins by nondenaturing polyacrylamide gel electrophoresis and affinity blotting onto nitrocellulose.

A latent endoribonuclease, RNase L, binds to and is activated by (2'-5')oligoadenylates ((2'-5')(A)n, n = 2-15). Binding to a labeled derivative of (2'-5')(A)n, [32P](2'-5')(A)3pCp, is detected as a protein-ligand complex observed following nondenaturing polyacrylamide gel electrophoresis. One major binding complex and two minor binding complexes are readily seen in cytoplasmic extracts from Ehrlich ascites tumor cells, murine tissue extracts and rabbit liver tissue extracts. At least one of the more rapidly migrating complexes appears to be a proteolytic degradation product of the larger [32P](2'-5')(A)3pCp binding protein. Cell and tissue extracts containing [32P](2'-5')(A)3pCp binding activity can be immobilized onto nitrocellulose filters and [32P](2'-5')(A)3pCp binding activity detected using a simple, rapid, economical affinity blot assay. Detection of [32P](2'-5')(A)3pCp binding proteins following electrophoresis on nondenaturing polyacrylamide gels and the affinity blot assay significantly improve and simplify the analysis of (2'-5')(A)n binding proteins.

Adenine Nucleotides↗
PubMed1992

A zymographic assay for detection of hyaluronidase activity on polyacrylamide gels and its application to enzymatic activity found in bacteria.

A zymographic assay for the determination of hyaluronidase activity in cell-free extracts on native polyacrylamide gels has been developed. In this assay an agarose replica of the polyacrylamide gel which contains hyaluronic acid and bovine serum albumin (BSA) was used. After an incubation at 37 degrees C to allow transfer and development of enzymatic activity, the hyaluronic acid and BSA were precipitated in the agarose gel with 2 M acetic acid. Areas of enzymatic activity appeared as clear zones in the agarose replica. The assay was sensitive and was used to demonstrate hyaluronidase activity in cell-free extracts from a number of bacterial and mammalian species.

Animals↗
PubMed1988

Renaturation of phosphorylase kinase activity from sodium dodecyl sulfate-polyacrylamide gels.

Phosphorylase kinase activity is renatured and detected in situ following electrophoresis of the denatured holoenzyme in a sodium dodecyl sulfate-polyacrylamide gel containing phosphorylase b that has been included in the gel polymerization according to the method of R. L. Geahlen et al. [(1986) Anal. Biochem. 153, 151-158]. Among the enzyme's four subunits, only gamma is catalytically active. When extract of rabbit muscle is electrophoresed and renatured in a similar manner, the phosphorylase-conversion activity is also associated only with a protein band that comigrates with the gamma subunit of phosphorylase kinase. This suggests that the gamma subunit of phosphorylase kinase may be the sole activity in rabbit muscle responsible for the phosphorylation of phosphorylase b. In an alternative method for the renaturation of activity from conventional sodium dodecyl sulfate-polyacrylamide gels, the subunits of the enzyme are visualized using 2.5 M KCl, excised from the gel, and eluted by diffusion into buffer containing sodium dodecyl sulfate, which is subsequently removed by acetone precipitation of the eluted subunits. Catalytic activity is recovered when the acetone precipitate of the extracted gamma subunit is dissolved in 6 M guanidine hydrochloride and diluted 50-fold into an activity assay. Inclusion of eluted alpha and beta subunits in the assay inhibits the activity of the gamma subunit, which supports our previous finding that the alpha and/or beta subunits suppress the activity of the catalytic gamma subunit [H. K. Paudel and G. M. Carlson (1987) J. Biol. Chem. 262, 11912-11915].

Adenosine Triphosphate↗
PubMed1975

Quantitative immunoelectrophoresis of proteins in human erythrocyte membranes. Analysis of protein bands obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. We have defined conditions that permit quantitative immunoelectrophoresis in agarose gels of dodecyl sulfate-solubilized erythrocyte membrane proteins. 2. Using human serum albumin, transferrin, MN-glycoprotein (glycophorin) and crude spectrin as test proteins, we found that accurate analyses are possible if samples and gels are 1% in non-ionic detergent (Berol EMU-043) or Triton X-100) and if no more than 100 nmol free dodecyl sulfate is applied per sample. 3. Dodecyl sulfate treated membranes analyzed by crossed immunoelectrophoresis using rabbit antibodies against membrane material yielded optimal precipitation patterns in gels containing 1% of non-ionic detergent. 4. Crossed immunoelectrophoresis in the presence of 1% of Berol revealed precipitates when 10 protein bands defined and isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis were run against anti-membrane antibodies. Seven of these bands showed more than one precipitation arc, indicating the presence of more than one antigenic component. 5. Crossed-line immunoelectrophoresis showed that dodecyl sulfate-polyacrylamide gel electrophoresis bands 1, 2 and 2.1 shared common antigenic components. The MN-glycoprotein was present in bands 3, 4A, 4B and 5, where antigenic components of the major intrinsic erythrocyte membrane protein, band 3, were also found. 6. After absorption of the anti-membrane antibody with intact erythrocytes, immunoelectrophoresis showed the disappearance of the MN-glycoprotein precipitates. An increase in the area below the precipitate corresponding to the major intrinsic protein (band 3) was also observed, indicating exposure of some antigens of this protein on the outer surface of intact cells. 7. After absorption of the antibody preparation with washed erythrocyte membranes, immunoprecipitates were not seen in any experiments, indicating that all antigenic determinants observed are exposed at one or both surfaces of the membrane. 8. Our analyses indicate that the peptide moieties of serum lipoproteins do not constitute a significant component of erythrocyte membranes.

Antibody Specificity↗
PubMed1978

Preparative two-dimensional polyacrylamide gel electrophoresis of rat liver ribosomal proteins and determination of their amino acid compositions.

1. By enlarging the dimensions of the gels used in the usual analytical two-dimensional polyacrylamide gel electrophoresis it is possible to separate much larger amounts of ribosomal protein in comparison to analytical separations. 15 mg of protein mixture of small or large subunits of rat liver ribosomes can be separated by this procedure. 2. The positions of the proteins in the two-dimensional patterns are identified with a special staining procedure. The proteins are eluted from the gels with SDS/phosphate buffers. 3. The purity of the extracted proteins was tested by one-dimensional SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis, respectively. 24 proteins of the small and 24 proteins of the large ribosomal subunit were isolated in pure form. 4. The amino acid compositions of 24 proteins of the small and of 19 proteins of the large subunit were determined.

Amino Acids↗
PubMed1976

The estimation and comparison of molecular weight of angiotensin I converting enzyme by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis.

The angiotensin I converting enzyme from rat lung was observed to be a glycoprotein containing 8.3% carbohydrate and consisting of a single polypeptide chain with an estimated molecular weight of 139 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and 150 000 by sucrose density gradient sedimentation analysis. A comparison of the mobility of angiotensin I converting enzyme from rat lung, rabbit lung, and two hog lung sources on sodium dodecyl sulfate-polyacrylamide gels indicates that all four enzymes have very similar molecular weights and subunit structures. Some previously reported molecular weight discrepancies appear to be due to anomalous behavior of the enzyme of gel filtration.

Animals↗
PubMed1979

Isolation and partial characterization of a guinea pig serum apolipoprotein comigrating with apo-E on sodium dodecyl sulfate-polyacrylamide electropherograms.

Study of guinea pig plasma lipoproteins has shown that they contain a polypeptide that comigrates with the arginine-rich polypeptide (apo-E) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This comigrating polypeptide differs from apo-E in its amino acid composition, immunological cross-reactivities, electrophoretic mobility in urea polyacrylamide gel, and elution volume from Sephadex gel columns. It is present in very low density lipoproteins and low density lipoproteins from both control and cholesterol-fed guinea pigs.

Amino Acids↗
PubMed1980

A two-dimensional polyacrylamide gel electrophoresis system for the analysis of mammalian cell surface proteins.

A detailed analysis of mammalian cell surface proteins is described by a new two-dimensional polyacrylamide gel electrophoresis technique. The first dimension gel contains 2% acrylamide, 0.1% sodium dodecyl sulfate, 0.3% Triton CF10 and 9 M urea. A combination of the detergents and urea permits the separation of poorly soluble, hydrophobic cell surface proteins. Under these conditions, the molecular size of proteins has a limited contribution to the fianl separation due to a low acrylamide concentration. Differences in charge properties, hydrophobicity, and glycosylation are the elements determining the resolution. In the second dimension, the proteins are separated primarily according to molecular weights, by a conventional polyacrylamide gel system in the presence of 0.1% sodium dodecyl sulfate. In this study, proteins of C6 rat glioma cell line are characterized. Cell surface proteins are specifically radio-labeled with 125I by a lactoperoxidase method, and compared with presumptive integral surface proteins which are resistant to extraction with 0.1 M NaOH. Also studied are total cellular proteins, fucose- and glucosamine-containing glycoproteins, and protein species with variable susceptibility to weak trypsin digestion. The electrophoresis system allows an unambiguous identification of each protein species.

Animals↗
PubMed1984

Two-dimensional peptide mapping by polyacrylamide-gel electrophoresis with limited proteolysis in SDS.

The peptide mapping method described by Cleveland, et al. (1) was improved to a two-dimensional analysis applicable to minute amounts (less than 0.5 microgram) of proteins. Radioiodinated proteins for analysis were purified by electrophoretic elution of the proteins from polyacrylamide gels into buffer containing 0.1% sodium dodecyl sulfate. The proteins were digested enzymatically in the presence of 0.1% sodium dodecyl sulfate and an excess of nonlabeled bovine serum albumin (0.2 mg/ml) relative to labeled substrate in order to attain reproducibility by maintaining a consistent substrate concentration among different samples. The peptides of these limited proteolytic products were resolved by two-dimensional polyacrylamide gel electrophoresis (isoelectric focusing followed by SDS-gels). The resulting 2D-peptide maps of murine and bovine albumin and a murine lymphocyte membrane protein, Tp100, showed excellent resolution and reproducibility.

Animals↗
PubMed1985

Protein blotting through a detergent layer, a simple method for detecting integral membrane proteins separated by SDS-polyacrylamide gel electrophoresis.

To selectively detect amphiphilic proteins from a mixture of proteins separated by SDS-polyacrylamide gel electrophoresis, the gel was electro-blotted through another polyacrylamide gel containing a non-ionic detergent (NP40) onto a nylon membrane filter. Most soluble proteins of E. coli passed through the detergent-containing gel, whereas a major fraction of the proteins from the cytoplasmic (inner) membrane, including the lactose and melibiose carrier proteins, were trapped in the detergent layer. The major outer membrane proteins, OmpA, OmpF and LamB, partitioned to the detergent layer only when solubilized at low temperature which avoids complete denaturation. This simple procedure, termed "detergent blotting", should have wide application in the study of integral membrane proteins.

Bacterial Outer Membrane Proteins↗
PubMed1990

Internal amino acid sequencing of proteins by in situ cyanogen bromide cleavage in polyacrylamide gels.

A new method was developed for generating peptide fragments for amino acid sequence analysis from polyacrylamide-gel separated proteins. This method involves in situ CNBr treatment of proteins in the polyacrylamide gel after their separation by electrophoresis. Pure CNBr peptides were recovered either by solvent extraction followed by microbore column reversed-phase HPLC or, alternatively, by a second electrophoretic separation step (SDS-PAGE) followed by electrotransfer of the peptides onto polyvinylidene difluoride (PVDF) membranes. These approaches yielded sequence data at subnanomole levels for a wide range of CNBr fragments recovered from gel-separated proteins.

Amino Acid Sequence↗
PubMed1977

Measurement of estradiol receptors in human breast tumors by polyacrylamide gel electrophoresis.

1. Specific estradiol receptors in cytosols from human breast tumors were measured by discontinuous polyacrylamide gel electrophoresis. 2. Binding of estradiol to contaminating sex hormone binding globulin was clearly separated from that due to the specific receptor. 3. The amount of estradiol bound by the receptor was linear with respect to the amount of protein added to gel. 4. The estradiol receptor peak of radioactivity showed saturability and specificity characteristic of an intracellular receptor. 5. The results show that polyacrylamide gel electrophoresis can be used to quantitate specific estradiol receptors in the presence of other high affinity binding components.

Breast Neoplasms↗
PubMed1977

Polyacrylamide gel electrophoresis of human erythrocyte membrane enzymes solubilized with triton X-100.

Human erythrocyte ghosts were solubilized in a low ionic strength medium containing 1% Triton X-100 and subjected to electrophoresis in polyacrylamide gels containing Triton X-100. Five major bands were stained with Coomassie Blue, all except one band being heterogenous when re-electrophoresed in gels containing sodium dodecyl sulphate. It was possible to detect acetylcholinesterase, non-specific esterase, ATPase, alkaline phosphatase, 5'-nucleotidase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and aldolase activities on the Triton-containing polyacrylamide gels. Two of the enzymes, ATPase and 5'-nucleotidase, showed substantial inhibition by Triton X-100 in quantitative studies. This appears to be a useful method for studying membrane enzymes in normal and pathological red cells.

Blood Proteins↗
PubMed1977

An accurate determination of human growth hormone content in different pituitary extracts, using a radioimmunoassay with polyacrylamide gel electrophoresis as a bound-free separation system.

Human growth hormone was extrated and purified according to the method of Roos et al. (Roos, P., Fevold, H.R. and Gemzell, C.A. (1963) Biochim. Biophys. Acta 74, 525). A first control of its purification and integrity was performed through molecular weight determination by gel filtration on Sephadex G-100 and on polyacrylamide gel electrophoresis (PAGE). Its biological activity was confirmed by the growth promoted in non-hypophysectomized rats at plateau. The main object, however, was the setting up of accurate, reproducible method tha could furnish the more absolute and comparable values of radioimmunoassayable HGH content in perfect agreement with the results obtained by other laboratories. This was accomplished through a radioimmunoassay system that uses HGH labelled with 125I, where separation of the bound from the free antigen is achieved on polyacrylamide gel electrophoresis, by a modification introduced in the original method of Davis. The resulting values, extremely close to that stated by the KABI-Laboratories (Stockolm), though obtained in quite different conditions of incubation, antibody concentration and with no use of second antibody, represent a confident approach to a comparable measure of this hormone in extracts, which can also be applied to plasma determinations.

Animals↗
PubMed1980

Cholesterol gas-liquid chromatographic microassay in serum lipoproteins separated by polyacrylamide gel electrophoresis.

A new method of cholesterol assay in serum lipoprotein fractions separated by electrophoresis on polyacrylamide plates is described: each lipoprotein fraction was collected by cutting the gel and, after gel dissolution, cholesterol was extracted and assayed by gas-liquid chromatography. This method associates the specificity and sensitivity of gas-liquid chromatography with the resolution of polyacrylamide gel electrophoresis, a highly reliable technique for hyperlipoproteinemia phenotyping. It is precise (variation coefficient below 2%), fast and needs only 2.5 microlitres of serum. Direct assay of cholesterol is feasible not only on each of the three main fractions, very low density lipoproteins, low density lipoproteins and high density lipoproteins, but also on intermediary fractions such as Lp(a) lipoprotein. This method should allow a better evaluation of the relationship between the serum cholesterol fractions and development of atherosclerosis.

Adolescent↗
PubMed1986

A map of urine proteins based on one-dimensional SDS-polyacrylamide gel electrophoresis and Western blotting using one microliter of unconcentrated urine.

A sensitive one-dimensional SDS-polyacrylamide gel electrophoretic system was devised whereby the proteins in 1 microliter of unconcentrated urine could be visualized by silver staining over the range 9,000-900,000 molecular weight. Identification of urine proteins was confirmed by Western blotting using peroxidase labelled antibodies. A map of the major proteins visualized in urine from individuals with renal disease was constructed. We conclude that the information available from the simple analysis of proteins according to their size is limited to general conclusions regarding whether proteinuria is likely to be of tubular or glomerular or mixed origin. More specific identification of individual proteins is not feasible because simple protein staining is not sufficiently reliable to identify individual proteins. The reasons for this conclusion are as follows: many proteins in urine migrate with similar apparent molecular weights, some proteins are not visualized by silver staining, and albumin polymeric complexes and fragments can be present at almost any molecular weight. However, one-dimensional SDS-polyacrylamide gel electrophoresis together with Western blotting does provide reliable information which might be clinically and experimentally useful.

Albuminuria↗