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Protein samples prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis are preferentially cleaved at aspartyl-prolyl peptide bonds upon heating at 110 degrees C. The presence of aspartyl-prolyl peptide bonds in a protein can therefore be detected by gel electrophoresis of heated samples and the resulting peptides mapped. The method of heat cleavage also works well with proteins in bands cut from electrophoresed gels using modified stacking conditions in the second electrophoresis. An immunoblotting procedure for peptide mapping of nanogram quantities of specific proteins in complex mixtures is demonstrated. Peptide maps produced by aspartyl-prolyl peptide bond cleavage of fructose-1,6-bisphosphatases from different sources show the effectiveness of the above techniques and suggest a conservation of aspartyl-prolyl peptide bonds in pig kidney and mouse and rat liver fructose-1,6-bisphosphatases.

Human platelets contain a polypeptide growth factor that stimulates the proliferation of connective tissue cells. Purification of this platelet-derived growth factor (PDGF) was accomplished by heat (100 degrees C) treatment of washed platelets and subsequent ion-exchange chromatography, gel filtration in 1 M acetic acid, isoelectric focusing, and preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis. PDGF has an isoelectric point of 9.8 and a molecular weight ranging from 13,000 to 16,000 as judged by gel filtration in 1 M acetic acid or analytical sodium dodecyl sulfate gel electrophoresis under reducing conditions. The specific activity of the purified PDGF is 20 million times greater than that found in unfractionated human serum. Purified PDGF stimulates replicative DNA synthesis and cell proliferation in quiescent density-arrested cultures of BALB/c 3T3 cells at concentrations of 1 ng/ml (0.1 nM).

A new agarose-acrylamide gradient slab gel electrophoresis system is described. The preparation of this new gel has been facilitated by the use of agarose with a relatively low gelation temperature. Fractionation of marker proteins and crosslinked proteins from a subcellular cytoskeletal preparation on agarose-acrylamide gradient gels is compared to that found using other acrylamide gel electrophoresis systems.

In two-dimensional denaturing gradient gel electrophoresis, DNA is digested with a restriction endonuclease and the resulting DNA fragments are separated as a function of size by conventional agarose gel electrophoresis. Following this first dimension electrophoresis, the fragment distribution is placed at the top of a denaturing gradient slab gel and electrophoresis is carried out parallel to the gradient direction. This second dimension separation is a complex function of the base sequence of each fragment. Analysis of the DNA fragment distribution as a function of fragment size allows the DNA size to be calculated. This method has been applied to calculate three microbial genome sizes: Mycoplasma capricolum, 724 kb; Acholeplasma laidlawii, 1646 kb; and Hemophilus influenzae, 1833 kb.

Electrophoretic separation of proteins in gels is a universally popular technique whose application is not, of course, restricted to membrane proteins. Nevertheless, the need in membrane protein research for techniques for the separation and analysis of complex mixtures of hydrophobic proteins has perhaps provided a major impetus for the development of improved electrophoretic methods over the last decade, particularly the separation of sodium dodecyl sulphate (SDS)—protein complexes on polyacrylamide gels. The latter technique, and the complementary use of isoelectric focusing methods (Chapter 2), are the basis of many aspects of membrane protein research. In view of this importance, I make no apology that this chapter describes electrophoretic methods which can equally be applied to soluble proteins. However, I have concentrated on those methods which have proven to be most useful for membrane proteins and highlighted particular problems which may be encountered in membrane research.

We describe an electrophoretic method for the transfer of macromolecules from polyacrylamide slab gels to ion-exchange paper without loss of clarity or resolution. A series of partial transfers provides numerous copies of a single gel separation, and these copies may be assayed independently with enzyme specific stains. This method therefore combines the advantages of polyacrylamide gel electrophoresis for detecting enzyme variation with an efficient method for examining numerous enzymes from a single gel separation.

A new method is described for specifically staining protein sulfhydryl groups after the proteins have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in slab gels. The stain will detect as little as 0.25 microgram of lysozyme and 1 microgram of most other proteins; the range of sensitivity for a specific protein depending on its sulfhydryl content. Proteins with no cysteine residues (type I collagen) and glycoproteins do not cause spurious staining.

Gel electrophoresis has been a powerful technique for the separation of peptides and proteins for many years. After electrophoresis separation on a polyacrylamide gel, the peptide bradykinin was localized using Coomassie Blue as a staining dye. Excess dye was removed by washing the gel with water. For mass spectrometric analysis, bands containing the peptide were crushed, extracted with acetic acid and the eluent applied to the fast-atom bombardment probe. Under these conditions the protonated molecule of bradykinin was clearly observed. Also apparent were sequence ions at about the same intensity observed from authentic bradykinin.

Using a combination of gel filtration and DEAE-cellulose chromatography, together with small-scale preparative polyacrylamide gel electrophoresis, we isolated five proteins (amelogenins) from demineralized bovine fetal dental enamel matrix. These purified proteins were characterized by amino acid analysis and gel electrophoresis. Comparisons of these data with those of other workers suggest that, although there are similarities in the published data between components of comparable electrophoretic mobility, there are gross differences in the reported amino acid compositions. It is suggested that these differences are due not to separative problems arising from reversible aggregations, but to inadequate comparisons of the electrophoretic and amino acid analytical data.

A method is described for the separation and purification of different molecular species of RNA in microquantities using polyacrylamide gel electrophoresis. The experimental procedure consists of the following steps; (i) partial prestaining of RNA with methylene blue at a concentration of the dye which would not affect the electrophoretic migration of the RNA bands, but which would permit visual observation of the migrating bands during electrophoresis; (ii) use of short columns just sufficient to achieve separation of each species of RNA as a single compact band rather than a series of bands; (iii) trapping of each of the eluting RNA species from the gel on DEAE-cellulose dises in sequential order; and (iv) elution of the RNAs from the DEAE-discs by extraction with triethylammonium bicarbonate and recovery of the corresponding RNAs by lyophylization.

An examination has been made of the effect which temperature of solubilization has upon the subsequent migration in SDS-polyacrylamide gel electrophoresis of proteins from the cell envelopes of Escherichia coli K12 and Neisseria sicca ATCC 9913. Conventional electrophoresis in tubes revealed substantial differences in the staining patterns of gels, depending upon whether the envelope samples were solubilized at 37 degrees C or 100 degrees C; in the case of N. sicca at least 6 of 13 discernible bands displayed heat-modifiable behavior. The relationship of the bands produced by each of the two temperatures was investigated by a two-dimensional electrophoresis procedure, in which a sample was solubilized at 37 degrees C and run in a usual cylindrical gel; the entire gel was then resolubilized at 100 degrees C, and laid along an acrylamide slab for electrophoresis in the second dimension. It was found that "free endotoxin" of both organisms examined contained the same major proteins as the total envelope fraction, and that these free endotoxin proteins showed the same heat-modifiable properties as when present in total envelopes.

Multidimensional electrophoresis (MD-analysis) is a technique for the high-resolution electrophoresis of proteins and polypeptides and for their characterization. It is presented here as three-dimensional electrophoresis for the separation and characterization of poliovirus-infected cells. This method is composed of three steps namely isoelectric focusing in urea-containing polyacrylamide gels (PAGIEF) as the first dimension, followed by electrophoresis of the gel piece from the first dimension in sodium dodecyl sulfate containing gels (SDS-PAGE) as the second dimension. Gel pieces from the two-dimensional gel subjected to limited proteolysis and subsequent SDS-PAGE of the peptides obtained constitute the third dimension. In addition, the influence of the most important parameters for the separation in the third dimension is demonstrated on serum albumins as substrates. The MD-analysis separates proteins and allows to characterize their relationship to each other and to identify unknown protein spots by comparison with peptide maps of known proteins.

The thyroxine-serum protein complexes have been studied by means of electrophoresis at pH 8.6 using starch gel and paper as a supporting medium. Borate buffer was used in the gel experiments and barbital or ammonium carbonate in the paper experiments. On gel electrophoresis, most humans bind thyroxine in and near the albumin zone. Band 1 corresponds to the fastest moving prealbumin, Band 2 is at the leading edge of the broad albumin zone, band 3 is diffuse and located in the slowest half of the albumin zone and Band 4 travels immediately behind the albumin. Monkeys have a pattern similar to humans but there is a variation in the position of Band 1 and in the fastest moving prealbumin protein which may constitute a polymorphic system. Concomitant variation was found in the mobility of the prealbumin thyroxine complex (TBPA) in paper electrophoresis in ammonium carbonate. In the cow, both Band 1 and TBPA appear to be absent, and other thjTOxine-protein bands in starch gel electrophoresis are found which hav...

Beta-N-acetylglucosaminidase in human parotid saliva was separated into two subfractions by diethylaminoethanol cellulose column chromatography. One subfraction of the enzyme was isolated and purified. Disk electrophoresis showed that the purified enzyme was homogeneous. The molecular weight of this enzyme was estimated to be about 153,000 by gel filtration and dodecyl sulfate polyacrylamide gel electrophoresis. N-acetyl-β-galactosaminides also were hydrolyzed by this enzyme at the same site.

Five reduced and alkylated proteins have been labeled with l-dimethylamino-naphthalene-5-sulfonyl (DNS) chloride in sodium dodecyl sulfate (SDS) solution. The fluorescent DNS-proteins have been subjected to disc gel electrophoresis. Sensitivity of the method is such that a few micrograms of labeled protein can be easily revealed on conventional gels by UV light. By reduction of the gel diameter to 1 mm, a minimum of 0.05 μg of protein can be detected. The DNS group has at most a minor effect on the migration of the protein in the SDS-disc gel electrophoresis system.

It has been found from a comparison of the electrophoretic mobilities on SDS-acrylamide gels of unmaleylated, maleylated, and demaleylated proteins that electrophoretic mobilities are affected by changes in charge as well as by changes in molecular size. Caution is therefore suggested in interpreting the values of molecular weight determined by electrophoresis in SDS-acrylamide gels.

The recovery of protein in preparative polyacrylamide gel electrophoresis (PAGE) is a function of protein load, buffer system (pH), and gel concentration. Recovery can be substantially improved (to about 90%) by polymerization of the gel in the presence of an extraneous protein (e.g., BSA, bacitracin) but not by addition of formamide, reducing agent, sucrose, or polyaminoacids to the gel.

EARLIER results1,2 suggested that chick interferon, purified by the methods of Fantes et al.1,3, was wholly or at least predominantly homogeneous, when judged by its behaviour on electrophoresis in polyacrylamide gel or in a sucrose gradient. The alkaline polyacrylamide gel electrophoresis method of Davis4 also suggested to us that the interferon was homogeneous2.

This study indicates that the precision of the RNA separation on the polyacrylamide gels is high, and separation of two species of RNA differing by 1 S unit is quite easily obtained. The relationships developed in this study were for only gel concentration and condition of electrophoresis. Further studies relating the Svedberg units and the migration distance with other variables such as gel concentration and conditions of electrophoresis could bring about a general equation which would greatly facilitate the identification of resolved species of RNA.

A new technique for the isolation of large (water-insoluble or slightly soluble) peptides is described. Peptides are separated by flat-bed electrophoresis on polyacrylamide gel in the presence of 6 M to 8 M urea, and stained with Amido black. The bands are cut out and extracted by soaking in 70% formic acid. Since the dye-peptide bond is a salt-linkage, the separation of peptide from the dye is easily achieved by passing through an anion-exchange resin. The technique permits electrophoresis at any desired pH and on a relatively large scale. An analytical adaptation useful for testing purity of isolated peptides involves sequential electrophoresis in two dimensions at two different pH values.

Cell extracts and culture filtrates were prepared from several mycobacterial species and subjected to two-dimensional acrylamide gel electrophoresis. The cell preparations were first electrophoresed in a 7% homogeneous gel followed by a second electrophoresis, at right angles to the first separation, in a 2 to 30% linear gel gradient slab. Because the stained slab resembled a “fingerprint” or “peptide map,” this procedure has been called “electroprotein mapping.” More than 200 protein-staining spots were detected in several of the cell extracts, each map being characteristic of the species examined. The single most characteristic portion of each map was the region between the 16 and 30% gel concentrations. This region consisted of several small-molecular-weight components (many of peptide proportions) which together appeared as the most anodic band in the 7% gel column (first separation). The number and location of these components in the linear gel slab (second separation) were specific for each species tested. By electrophoresis of molecular-weight markers (i.e., albumin, myoglobin, cytochrome c ) with and across (transverse electrophoresis) the gel gradient, the relative molecular weights of the unknown cell extract components could be estimated. These results suggest that two-dimensional acrylamide gel electrophoresis is a useful tool for the characterization of mycobacterial cell constituents.

THE investigation of the molecular weight and the electrophoretic behaviour of globins at pH. 2–4 has led to a better understanding of the structure of the haemoglobin molecule. At a pH less than 6 there is a progressive decrease of the molecular weight of human haemoglobin1. At pH 11 the value of the sedimentation coefficient is 2.55, which points to a molecular weight of about 33,000 instead of 67,000, a value normally found2.

The electrophoretic mobility of the two major RNA components of rat-liver mitochondria gradually decreases relative to the mobility of the cell-sap ribosomal RNA components when the temperature of electrophoresis is raised from 2 to 28°. Hence, the calculated apparent molecular weight of rat mitochondrial RNA is 40% higher at 28 than at 2°. This dependence of apparent molecular weight on the temperature of electrophoresis can explain the large differences in apparent molecular weight reported for the mitochondrial RNAs from different animals.

Double-stranded ribonucleic acid ( RNA ) extracted from purified reoviruses of all three serotypes and from type 3 virus-infected cells was analyzed by polyacrylamide gel electrophoresis. It was calculated that each RNA includes 10 segments: 3 large, 3 intermediate, and 4 small fragments corresponding to molecular weights of about 2.5, 1.4, and 0.8 × 10 6 daltons, respectively, or a total of 15 × 10 6 daltons.

Publisher Summary This chapter describes polyacrylamide gel electrophoresis of viral RNA. It explains that the application of polyacrylamide gel electrophoresis to the fractionation of viral RNA has added a powerful tool to the arsenal of the virologist. The electrophoretic mobility of RNA in free solution does not show marked size dependence. This is expected because there is no variation in the charge to mass ratio of RNA with changes in molecular weight. The use of a polyacrylamide gel support in zone electrophoresis of macromolecular substances takes advantage of the capacity of the gel to sieve these high molecular species and allows for the separation of species of identical free solution mobilities. The pore size of the gel matrix is chosen such that it approaches the molecular dimensions of migrating ions. The simplest application of polyacrylamide gel electrophoresis is in the study of the structural RNA species of different viruses. Recovery of RNA fractions from the gels for studies, such as primary sequencing, biological activity, and studies on sequence homologies by DNA-RNA hybridization is necessary for thorough understanding of viral physiology.

The apparatus for preparative polyacrylamide gel electrophoresis has been devised, by which eluted materials can be sampled continuously and quantitatively in a drop-scale.

Soluble antigen-antibody complexes between ovotransferrin and its antibody were separated by rate zonal centrifugation and by acrylamide gel electrophoresis in 5.0% gel or in a discontinuous gel gradient. The isolation of centrifuged fractions was facilitated by photopolymerizing the tube content in acrylamide gel. Both methods gave identical types of antigen-antibody complexes, characterized by electrophoretic mobility, antigen-antibody ration, and sedimentation constant. The acrylamide gel electrophoresis in 5.0% gel is particularly suitable for the identification and the quantitative determination of various Ag Ab complexes. Small quantities of concentrated Ag Ab complexes can be obtained easily in the “pore limit” electrophoresis, larger quantities by zonal centrifugation in acrylamide containing sucrose gradients.

Summary: Colicins can be separated by polyacrylamide gel electrophoresis, so allowing multiple colicinogeny to be detected with a universal indicator strain.

Highly purified J-chain was isolated from S-sulfonated human polymeric myeloma IgA by gel filtration and DEAE-cellulose chromatography, without using any dissociating agents. The purified J-chain was homogeneous upon immunoelectrophoresis and electrophoresis in agarose gel, alkaline-urea polyacrylamide gel, and sodium dodecyl sulfate polyacrylamide gel. The molecular weight of the J-chain, estimated by electrophoresis in sodium dodecyl sulfate polyacrylamide gel, was found to be 24 000 ± 1200 (S.D.). The J-chain dimerized in non-dissociating solvents, even in the presence of weak dissociating agents, such as 1 M propionic acid. During the isolation of the J-chain, two minor protein components which upon electrophoresis migrated, respectively, faster and slower than the J-chain itself were identified. These two components were antigenically unrelated to each other but both were clearly precipitated with anti-J-chain antisera, giving a reaction of partial identity to the J-chain. They were considered as two different fragments of J-chain, produced by some unknown protease(s).

Publisher Summary This chapter describes polyacrylamide gel electrophoresis of viral proteins. Viral structural and nonstructural proteins are agents of expression for the information carried in viral nucleic acid genomes. An understanding of the behavior of a virus requires its proteins be characterized. For this purpose and for the characterization of nucleic acids, electrophoresis in gels is the most sensitive and versatile method. The genetic content and number of proteins of most viruses is relatively small compared even to simple cells. Two aspects of gels as supporting media for zonal electrophoresis experiments are responsible for the dramatic separations. First, as simple anti-convection media gels, they are more homogeneous and stable than other commonly used materials. Second, with the correct choice of gel concentrations, the spaces in the gel matrix are of similar dimensions to macromolecules so that selective sieving occurs during the electrophoretic migration. Gels composed of polyacrylamide are extensively used at the present time.

SummaryStarch-gel electrophoresis has been employed to separate the proteins in human and rat brain. This technic yielded results superior to those attained by paper or agar-gel electrophoresis. Separation of water-soluble proteins on starch-gel yielded 11–12 discrete protein bands in rat brain and 14–15 bands in human white matter. Human white matter proteins are characterized by low concentration of albumin-type proteins.

A simple and rapid technique for the quantitative determination of hemoglobins separated by starch gel electrophoresis has been described. This method, in which the hemoglobin concentration is measured in an alkaline starch-hemoglobin solution, has been used to determine the per cent HbS in individuals with sickle cell trait.

A human-specific esterase was isolated from urine and partially characterized by ammonium sulfate precipitation, starch block electrophoresis, and gel filtration. Its molecular weight was estimated to be 136,000 by polyacrylamide gel electrophoresis in 0.17. SDS. A diffusion coefficient of 8.6 X 10−7 was determined by the L-plate method. Immunologic identity was shown between the urinary esterase and a human tissue esterase.

— The isolation of a perchloric acid-soluble low molecular weight protein from brain of Macaca irus is reported. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing indicate that the protein is free of impurities. The molecular weight, as determined by gel filtration and sodium dodecyl sulphate gel electrophoresis, is shown to be 10,400 and 9900, respectively. This is in agreement with the value of 10,700 obtained from amino acid analysis. The protein contains 27 per cent acid amino acids and 15 per cent basic amino acids. However, the relatively high amide content gives the protein a neutral nature as shown by isoelectric point determination using gel isoelectric focusing.

1. The tRNA (4S) of E. coli has been fractionated by electrophoresis on polyacrylamide gel and separated into two electrophoretic classes designated 4S RNA A and 4S RNA B.

The dependence of the mobility of RNA species on their concentration in agar gel electrophoresis is explained in terms of adsorption on the gel matrix. The number of interaction sites on RNA molecules depends on the origin of the RNA and on the extent of deproteinization. The ratio of sites on the RNA of the large and small ribosomal subunits is equal to the ratio of their molecular weights for both rat liver and E. coli . The results suggest that the interactions with the gel are due to some impurity, most probably protein.

The solubilization and electrophoresis of membrane proteins with phenol and acetic acid or urea usually have deleterious effects on enzyme activities. A method is reported of solubilizing rat-liver plasma membrane proteins, by the use of the detergents sodium dodecyl sulfate, sodium deoxycholate, and Triton X-100, for subsequent electrophoresis of the enzymes on 7% polyacrylamide gel. An important feature of the method is the incorporation of detergent into the gel and the electrophoresis buffer. By use of this procedure, non-specific esterases, an alkaline phosphatase, and alkaline phosphodiesterase are detectable on the gels. The method is compared with other techniques currently being used, and it would appear that this method will have general usefulness in the study of membrane enzymes.

Some of the electrophoretic parameters are examined with respect to their effect on restricting the amount of RNA analyzable per gel. Acrylamide concentration was found to be the most important. By addition of a dilute pregel to the analyzing gel, up to 0.8–1 mg RNA can be examined with excellent resolution on gels with diameter 6 mm, and up to 2.5 mg RNA on gels with diameter 10 mm. The suggested principle might also be used to increase the yield of preparative electrophoresis. The large amounts of RNA introduce special difficulties in the scanning of the ultraviolet absorption. Suggestions are made on how to overcome these difficulties.

Increased interest in histone fractionation is evidenced by the many published electrophoretic techniques using starch (1–3) and polyacrylamide (4–6) gels. Twenty-two components of calf thymus histone have been identified by Neelin and Neelin (7) with starch gel electrophoresis. Driedger et al. (5) have obtained 18 histone bands by acrylamide gel electrophoresis and even more by sequential polyacrylamide and starch gel electrophoresis. However, fractionation of the total histone by various electrophoretic techniques often fails to give distinct resolution of bands, possible due to the complexity of the patterns obtained. In addition, present methods involve extensive or elaborate preparation, e.g., starch gels prepared in 7 M urea are difficult to manipulate (7); polyacrylamide gel electrophoresis requires washing and soaking the gel for several days before use (5). This paper describes an electrophoretic method which fractionates histone into a number of discrete bands. The technique, in addition to offering good resolution of fractions, eliminates the necessity for elaborate gel preparation and handling. Especially good separations of the arginine-rich histones have been obtained.