Polyacrylamide Gel Electrophoresis System Research Articles

Enhanced resolution of circular and linear molecular forms of viroid and viroid-like RNA by electrophoresis in a discontinuous-pH system

A discontinuous-pH polyacrylamide gel electrophoresis system is described. An increase in the pH differential between the gel and the running buffer enhances the separation of low molecular weight circular and linear RNA molecules. Highly purified preparations of the circular form of viroids can be obtained with this procedure. Since all the linear RNAs of similar molecular weight migrate with the front, a relatively clean background can be obtained even when crude extracts are used. This facilitates an improved separation and identification of similarly sized viroid-like RNAs. The conditions of electrophoresis in low salt and 8 m urea also permit the effective transfer of RNA molecules directly to nylon-based membranes without any additional denaturation treatment.

Synthesis and secretion of a nerve growth-stimulating factor by neonatal mouse astrocyte cells in vitro.

Neonatal mouse astroglial cells cultured in a serum-free medium synthesize and secrete a trophic growth factor which resembles nerve growth factor (NGF). The NGF-like factor reacts with antiserum to beta subunit of NGF (beta-NGF) and, after labeling with [35S] cystine, migrates similarly to purified mouse beta-NGF in SDS polyacrylamide gel electrophoresis and Sephadex G-100 gel filtration systems. The astrocyte cell NGF-like factor displays beta-NGF-like neurite growth-promoting activity for the clonal rat pheochromocytoma (PC-12) cell line and this bioactivity is blocked by beta-NGF antiserum. These results indicate that NGF-like factor synthesized and secreted by astroglial cells, is similar, if not identical, to beta-NGF from the mouse submandibular gland and further support a potential role for NGF in the central nervous system.

Simple polycrylamide gel electrophoresis in continuous carbonate buffer system suitable for the analysis of ascitic fluids of hybridoma bearing mice

A convenient simple polyacrylamide gel electrophoresis system is described, suitable for semiquantitative analysis of the content of monoclonal antibodies in ascitic fluids of hybridoma-bearing mice.

Analysis of erythrocyte protein methyl esters by two-dimensional gel electrophoresis under acidic separating conditions

A two-dimensional polyacrylamide gel electrophoresis system which is suitable for the analysis of protein methylation reactions in cells incubated with l-[ methyl- 3H]methionine is described. The procedure separates proteins under primarily acidic conditions by isoelectric focusing in the first dimension and by sodium dodecyl sulfate electrophoresis at pH 2.4 in the second dimension. The low pH is essential for preserving protein [ 3H]methyl esters, but it limits the effective separating range of this system to proteins with isoelectric points between 4 and 8. With this system, we have shown that most, if not all, erythrocyte membrane and cytosolic proteins can act as substoichiometric methyl acceptors for an intracellular S-adenosylmethionine-dependent carboxyl methyltransferase and that protein carboxyl methylation reactions may be the major methyl transfer reaction in erythrocytes. These results are most consistent with the generation of protein substrate sites for the carboxyl methyltransferase by spontaneous deamidation and racemization reactions.

Variation in Estimates of Molecular Weights of Cereal Prolamins by SDS-PAGE

The total reduced, alcohol-soluble protein fractions from barley, wheat, rye, maize, millet and sorghum, which we term ‘prolamin’ fractions, were separated using three systems of Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and the relative molecular masses (Mrs) of the major components determined by comparison with the mobilities of standard proteins. Three types of errors were identified for prolamins of the Triticeae: over-estimation of Mrs compared with those determined by physical methods or from protein sequences, increases in Mrs due to the inclusion of urea in the gels and differences between Mrs determined, in the absence of urea, by different systems. Although different gel systems also gave different Mrs for some prolamins of the Panicoideae, the inclusion of urea had no effect. The results are discussed in relation to the amino acid compositions and secondary structures of the proteins.

Separation of gliadin at pH 3.1 in a polyacrylamide gel suitable for blotting procedures

A polyacrylamide gel electrophoresis system for the separation of gliadin at acidic pH is described. The gel is cast at neutral pH with polymerization in 20 min. Equilibrium of the gel to pH 3.1 takes place during the electrophoresis. The gel is highly uniform with good mechanical properties and therefore suitable for blotting procedures.

Optimization of polyacrylamide gel electrophoresis conditions used for sequencing mixed oligodeoxyribonucleotides.

We investigated two components of the polyacrylamide gel electrophoresis system used for sequencing DNA to improve the system for sequencing synthesized oligodeoxyribonucleotides containing positions of degeneracy. First, we varied the ratio of methylene-bis-acrylamide (MBA) to acrylamide from that commonly used in DNA sequencing gels (1% MBA:19% acrylamide). A moderate increase in the MBA:acrylamide ratio proves optimal when sequencing is used to confirm that a synthesized fragment contains equivalent stoichiometries of the different nucleotides at a given position of degeneracy. Such information is particularly important if the degenerate oligodeoxyribonucleotide preparation is to be employed as a hybridization probe. A further increase in the MBA:acrylamide ratio (3% MBA:19% acrylamide) produces a gel in which the sequence of an oligodeoxyribonucleotide mixture containing several positions of degeneracy can be read most easily. Increasing the MBA acrylamide ratio suppresses the effect of base composition on electrophoretic mobility of a fragment. Second, we investigated the use of a Tris-citrate buffer system in place of the standard Tris-borate system. We found the Tris-citrate system to be significantly more effective in preventing discontinuities in the banding pattern of the smaller fragments. Finally, we show that high MBA gels are also effective in resolving mixtures of oligoribonucleotides such as those produced by T1 ribonuclease digestion of small RNAs.

Purification and properties of rat uterine procollagenase

A procollagenase from monolayer cultures of postpartum rat uterine cells has been purified. The crucial step in the purification is the binding of the procollagenase from crude, fetal bovine serum-containing culture medium to heparin-Sepharose, followed by elution with extremely low concentrations (5–10 n m) of dextran sulfate. Resultant eluates contain 8–10% procollagenase. Purification is completed by ion-exchange chromatography on DEAE-Sepharose, gel filtration on AcA-44, and chromatography on blue-Sepharose. Rat uterine procollagenase appears as a protein doublet of M r ~ 58,000, as indicated by two polyacrylamide gel electrophoresis systems, by AcA-44 chromatography, and by equilibrium sedimentation ultracentrifugal analysis. The proenzyme forms are converted by trypsin to an active enzyme doublet of M r ~ 48,000. Small amounts of active enzyme, which are often generated during the purification, are electrophoretically indistinguishable from trypsin-activated collagenase. Active collagenase can be separated from the zymogen forms by DEAE-Sepharose chromatography. The two forms of the proenzyme doublet can be partially separated by gel filtration on AcA-44 and preliminary analysis indicates each has equal collagenolytic activity. The amino acid analysis of rat uterine collagenase reveals it to be markedly different from two other vertebrate collagenases whose composition is known. The uterine proenzyme is unusually rich in glycine and in the hydroxy amino acids and is considerably more acidic than the human skin fibroblast collagenase, consistent with the different ionexchange behavior of the two molecules. The specific activity of rat uterine collagenase at 37 °C is approximately 3000 μg collagen/min/mg, using native reconstituted guinea pig skin type I collagen fibrils as substrate. The enzyme cleaves denatured collagen, but fails to attack a variety of noncollagen proteins.

Yeast ribosomal proteins: Electrophoretic analysis in four two-dimensional gel systems—Correlation of nomenclatures

Proteins from the yeast Saccharomyces cerevisiae 40S and 60S ribosomal subunits were analyzed in four mutually related two-dimensional polyacrylamide gel electrophoresis systems. The position of every protein was determined in each system using the “four-corners” method. Two of the four gels used incorporate conditions common to various analytical systems used in other laboratories. The numbering of the proteins in each system is similar to the standard nomenclature for yeast ribosomal proteins proposed by Bollen, Mager and Planta [Molec Biol Rep (1981) 8: 37–44]. The “four corners” analysis facilitates the correlation of other data resulting from the use of different analytical and numbering systems and obviates occasional erroneous designations and discrepancies in work published on yeast ribosomal proteins.

Alkaline Phosphatase Purification: A Comparison of Prep-PAGE with Cyclic Processes

Abstract Enzyme purification has been investigated in a preparative-scale polyacrylamide gel electrophoresis system, the Buchler Poly-Prep 200. Experimental results are presented for the optimization of this process using human placental alkaline phosphatase as a typical enzyme. The variables considered are electric field strength, feed concentration, buffer ionic strength, and buffer pH. Enzymes may also be purified by adsorption/desorption onto an ion exchange resin such as DEAE Sepharose in either of two cyclic processes-parametric pumping or cycling zone adsorption. Comparison of the three processes indicates that polyacrylamide gel electrophoresis has the highest purification factor and the greatest enzyme activity recovered, but also the lowest rate of production.

Use of benzyldimethyl- n-hexadecylammonium chloride (“16-BAC”), a cationic detergent, in an acidic polyacrylamide gel electrophoresis system to detect base labile protein methylation in intact cells

A discontinuous polyacrylamide gel system operating at pH 4.0–1.5 which resolves proteins bearing base labile groups extracted from intact cells is described. It uses potassium phosphate buffer in the running and stacking gel and glycine as the trailing ion component. Proteins are solubilized with urea and benzyldimethyl- n-hexadecylammonium chloride, a cationic detergent. The utility of the system is illustrated by fluorographs of the pattern of protein methylation in blood platelets and the HL60 promyelocyte cell line.

Reinvestigation of the chlorophyll distribution among the chlorophyll-proteins and chlorophyll-protein complexes of Hordeum vulgare L.

Solubilization of barley (Hordeum vulgare L.) thylakoid membranes with sodium dodecylsulphate plus sodium deoxycholate with or without Triton X-100 and subsequent fractionation in the polyacrylamide gel electrophoresis system described in this paper resulted: (1) in the resolution of the chlorophyll-proteins and chlorophyll-protein complexes commonly known as CP1a, CP1, LHCP(1), LHCP(2), CPa and LHCP(3); (2) in the highly increased stability of CP1 and CP1a, as judged by their chlorophyll content, (3) at the expense of the free pigment concentration (4) which could be reduced to a negligible amount. Some 40% of the total chlorophyll contained in the mature higher plant thylakoid membrane is associated with CP1 and CP1a and as already suggested before [19] no significant amount of free chlorophyll occurs in vivo.

Characterization of the human plasma binding protein for vitamin D and its metabolites synthesized by the human hepatoma-derived cell line, Hep 3B.

Screening of three human hepatoma-derived cell lines revealed the presence of an immunologically similar plasma binding protein for vitamin D and its metabolites in media from Hep 3B cells. Approximately 3% of protein synthesized and secreted by these cells was immunoprecipitated by specific antiserum to the D-binding protein. Medium content of the protein increased over 11 days following cell seeding, and negligible amounts of 125I-labeled binding protein added to cultures were degraded over 48 h. The hepatoma-derived binding protein was indistinguishable from plasma binding protein or reference pure protein in gel filtration, sucrose gradient ultracentrifugation, and polyacrylamide gel electrophoresis systems. The Hep 3B cell product was found to bind mole/mol with monomeric actin, and bind vitamin D sterols with an affinity and specificity characteristic of the human plasma binding protein. The findings argue strongly for the identity of the Hep 3B cell product and the human plasma protein. The continuous availability of the Hep 3B cell line provides a reasonable model for investigations of biosynthesis and release of this important plasma protein.

Improved methodology for analysis and quantitation of proteins on one-dimensional silver-stained slab gels

A sodium dodecyl sulfate-discontinuous polyacrylamide gel electrophoresis system for separation and quantitation of low-molecular-weight (75 to 10K Da) proteins from single muscle fibers is described. Slab gels (0.75 mm thick) were stained using an improved silver-stain technique which does not require photographic fixers in order to achieve low-level background staining. The modified staining procedure uses continuous flow washing to minimize the handling of gels. The procedure has high sensitivity and gave a linear response between approximately 2 and 70 ng of protein per band. In addition, a convenient method for mounting slab gels for photography, scanning, and long-term storage has been developed.

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells.

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells.

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

18] Slab gel system for the resolution of oligopeptides below molecular weight of 10,000

Publisher Summary The systems of polyacrylamide gel electrophoresis that are in current use for the molecular weight determinations of conventional proteins are not well suited for resolving proteins below 10,000. This chapter describes certain modifications of the basic Swank and Munkres procedure that allow the gels to be slab-cast. The method is favorable, particularly for the resolution of small oligopeptides that are between Mr 1500 and 10,000. In preparing the gel, add everything except the SDS, TEMED, and ammonium persulfate to a small beaker; place the beaker in a 50-55° C water bath, and swirl gently until solid material is completely dissolved. After the gel is mounted on the gel apparatus and the buffer added, the sample wells must be washed out thoroughly just before application of the samples. Excess urea must be removed if the gel is to be dried back for autoradiography without prior staining or if it is to be treated for fluorography.

Reinvestigation of the chlorophyll distribution among the chlorophyll-proteins and chlorophyll-protein complexes of Hordeum vulgare L.

Solubilization of barley (Hordeum vulgare L.) thylakoid membranes with sodium dodecylsulphate plus sodium deoxycholate with or without Triton X-100 and subsequent fractionation in the polyacrylamide gel electrophoresis system described in this paper resulted: (1) in the resolution of the chlorophyll-proteins and chlorophyll-protein complexes commonly known as CP1a, CP1, LHCP(1), LHCP(2), CPa and LHCP(3); (2) in the highly increased stability of CP1 and CP1a, as judged by their chlorophyll content, (3) at the expense of the free pigment concentration (4) which could be reduced to a negligible amount. Some 40% of the total chlorophyll contained in the mature higher plant thylakoid membrane is associated with CP1 and CP1 a and as already suggested before [19] no significant amount of free chlorophyll occurs in vivo.

A new polyacrylamide gel electrophoresis system. Separation of small peptides and proteins in a volatile buffer system after modification with a strongly acidic fluorescent NH2 reagent.

Polyacrylamide gel electrophoresis is one of the most efficient methods for separation and identification of proteins. A new gel electrophoresis system has been developed in order to facilitate both separation and extraction of peptides and proteins ranging in molecular weight from approximately 200 up to 100 000. This system involves the use of a volatile buffer, triethylamine/formic acid pH 11.7, and a reaction with a covalently binding NH2 reagent, 1,3,6-trisulfonylpyrene 8-isothiocyanate. Under these conditions, the addition of strongly negative charges to proteins is achieved which allows migration according to molecular weight. The modified proteins being fluorescent require neither fixation nor staining for their detection. This is an absolute requirement especially when dealing with small peptides. An additional advantage of such a modification is the increased solubility of the proteins which makes their extraction easier and more efficient. THe extracted proteins are easily freed from salts and other small molecules simply by evaporation and they are readily available for sequencing analysis using Edman degradation, carboxypeptidase digestion or amino acid analysis.

Messenger RNA encoding basic chromosomal proteins of mouse testis

Considerable knowledge has accumulated concerning histones and other basic chromosomal proteins which appear in mammalian male germ cells during spermatogenesis. The best characterized of these proteins are testis protein (TP) and protamine. Both are small and highly basic, TP being present in early spermatids while two molecular species of mouse protamine may be detected in spermatids in the later stages of differentiation and in sperm. The amino acid sequence of TP from rat and of a single species of protamine from bull have been reported. In contrast to this detailed information related to protein structure, relatively little is known of the mechanisms involved in synthesis of the proteins or of the controls which regulate their synthesis during spermatogenesis. In the study reported here, messenger RNA (mRNA) from mouse testis was examined for its ability to act as a template for in vitro synthesis of TP and protamine. Three polyacrylamide gel electrophoresis systems were developed to distinguish TP and the two species of mouse protamine among in vitro translation products synthesized by a cell-free wheat germ extract. A 6 S RNA encoding TP was identified by translation of sucrose gradient fractions. RNA encoding TP was also found among the testis RNAs which were bound to oligo(dT)-cellulose during affinity chromatography. These physical properties and apparent abundance of this TP mRNA suggest that it may be a useful probe for further studies of RNA metabolism and gene regulation. In no case was an in vitro translation product comigrating with the two protamine species observed, suggesting the possibility that protamine may be synthesized as a precursor and later processed to the species which are found in sperm.