Two-dimensional Gel Electrophoresis Method Research Articles

Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family.

A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows one to estimate the number of hybridizing genes contained in each large DNA fragment. We have also developed a novel method to increase the separation, resolution and hybridization signal in the second dimension by condensing the bands from the first dimension into spots. As an example, we have applied these techniques to determine the organization of the murine T-cell receptor gamma locus. The murine gamma gene family was found to be contained on two DNA fragments encompassing 195 kilobases of DNA. The two-dimensional gel electrophoresis method is particularly useful in the analysis of the organization of multigenic families where single copy probes are not readily available, and should extend the potential usefulness of field inversion gel electrophoresis in gene mapping.

Further studies of the plasma alpha 1 B-glycoprotein polymorphism: two new alleles and allele frequencies in Caucasians and in American blacks.

Two new alleles (A1 B*3 and A1 B*4) of human plasma alpha 1 B-glycoprotein (alpha 1 B) were reported. alpha 1 B phenotyping was done by using either a simple method of two-dimensional (2-D) agarose gel-horizontal polyacrylamide gel electrophoresis (PAGE) followed by protein staining or by one-dimensional horizontal PAGE and immunoblotting. Seven different alpha 1 B phenotypes (1-1, 1-2, 1-3, 1-4, 2-2, 2-3 and 3-3) were observed; phenotypes 1-3 and 1-4 were differentiated from each other only by the 2-D method. The respective frequencies Af A1 B*1, A1 B*2, A1 B*3 and A1 B*4 alleles in the studied populations were estimated as follows: American Blacks (New York) 0.732, 0.204, 0.064, 0; American Whites (New York) 0.947, 0.053; Czechs (Mĕlník) 0.964, 0.034, 0, 0.002; Slovaks (Bratislava and Trencin) 0.977, 0.023, 0, 0. The population of American Blacks showed a much higher degree of alpha 1 B polymorphism (polymorphism information content = 0.37) than the Caucasian populations that have been studied.

Stability of protein and mRNA in human postmortem liver-analysis by two-dimensional gel electrophoresis

The method of two-dimensional polyacrylamide gel electrophoresis has been used to investigate the post mortem stability of protein and mRNA in human liver. The electrophoretic mobility of proteins and of the in vitro translation products of the mRNA were found to be essentially unaffected by incubation of the liver at 37° C for up to 2 h or at 4°C for up to 16 h. This study indicates that the major protein and mRNA species in liver are stable enough following death to allow meaningful studies on tissue collected under standard autopsy conditions.

Modulation of yeast 5 S rRNA synthesis in vitro by ribosomal protein YL3. A possible regulatory loop.

Synthesis of yeast 5 S rRNA in a homologous cell-free system has previously been shown to be inhibited by exogenous yeast 5 S rRNA. This inhibition is diminished when 5 S rDNA is first incubated with the cell-free system, implying the presence of a transcription factor IIIA analog in yeast, and is dependent on the nucleotide sequence of the 5 S rRNA added. A two-dimensional gel electrophoresis method was used to demonstrate assembly of newly synthesized 5 S rRNA into a ribonucleoprotein complex containing ribosomal protein YL3. Immunodepletion of the cell-free system with anti-YL3 IgG was found to increase the sensitivity of 5 S rRNA synthesis to inhibition by 5 S rRNA. We propose a model for the regulation of yeast 5 S rRNA synthesis in which YL3 competes with the presumptive transcription factor IIIA analog for binding of 5 S rRNA.

Genetic polymorphism of human plasma alpha 1B-glycoprotein: phenotyping by immunoblotting or by a simple method of 2-D electrophoresis.

Genetic polymorphism of human plasma (serum) alpha 1B-glycoprotein (alpha 1B) was observed using one-dimensional horizontal polyacrylamide gel electrophoresis (PAGE) pH 9.0 of plasma samples followed by Western blotting with specific antiserum to alpha 1B. A simple method of two-dimensional agarose gel electrophoresis (pH 5.4)-horizontal PAGE (pH 9.0) of plasma samples, followed by general protein staining, was reported as an alternative method for alpha 1B typing. The three different phenotypes of alpha 1B observed (designated 1-1, 1-2, and 2-2) were apparently identical to those reported by Altland et al. (1983), who used double one-dimensional electrophoresis. Family data supported the hypothesis that the three alpha 1B phenotypes are determined by two codominant alleles at an autosomal locus, designated A1B. Allele frequencies in a Swedish population were: A1B1, 0.937; A1B2, 0.063; PIC, 0.111. For clues on linkage relationships of human A1B, the previously known linkages of A1B in pigs and horses, including the one between A1B and the gene that determines susceptibility to malignant hyperthermia in pigs were discussed.

Primary structures of and genes for new ribosomal proteins A and B in Escherichia coli.

We determined the partial primary structures of and identified the genes for new basic proteins A and B in Escherichia coli ribosomal 50S subunits, found by means of an improved two-dimensional gel electrophoresis method. The sequence up to the 17th amino acid of protein B was in agreement with that of the X gene in the spc operon. The gene for protein A was searched for in the GenBank data base using the sequence up to the 35th amino acid, and was found at a locus between infC and rplT. The base sequence indicated that protein A contained 64 amino acids and had a molecular weight of 6,984. We conclude that proteins A and B are intrinsic ribosomal proteins, and propose calling their genes, rpmI and rpmJ, respectively.

Two-dimensional gel electrophoresis of proteins for genetic studies in Douglas fir (Pseudotsuga menziesii).

A method of two-dimensional gel electrophoresis of proteins from Douglas fir needles is described. Extraction in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol followed by heating at 100 degrees C produces reliable two-dimensional gels which are convenient for genetic studies. Three genotypes from different geographical origins have been compared: among 225 loci expressed, 22 display regulatory variations and 7 show allelic variations. Thus it is now possible to undertake the genetic study of Douglas fir using this powerful technique.

Myosin components of the latissimus dorsi and the pectoralis major muscles in the dystrophic chicken.

The myosin composition of the anterior latissimus dorsi, the posterior latissimus dorsi, and the pectoralis major muscles was examined in the inbred White Leghorn dystrophic chicken and its isogenic normal line at different ages during development and maturation. Using the biochemical methods of native gel electrophoresis, one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), and peptide mapping, it was found that myosin isozyme changes occurred normally in the anterior latissimus dorsi muscle. However, in the posterior latissimus dorsi muscle, slow myosin components which were not present in the adult normal muscle were present in the adult dystrophic muscle. In addition, the pectoralis major muscle of the dystrophic chicken failed to undergo the neonatal to adult fast myosin isozyme transition. Our data also showed that muscle cell cultures derived from the pectoralis major muscle of dystrophic chickens expressed identical myosin components to cultures derived from normal embryos. However, since these cultures only produced embryonic myosins even after 1 month in culture, it implied that cells in tissue culture were phenotypically normal because present cell culture conditions were insufficient to induce the fetal to adult isozyme changes.

Mammalian beta-adrenergic receptors. Structural differences in beta 1 and beta 2 subtypes revealed by peptide maps.

Photoaffinity labeling techniques using p-azido-m-[125I]iodobenzylcarazolol have recently demonstrated that both the beta 1- and beta 2-adrenergic receptor-binding subunits from mammalian tissues including heart, lung, and erythrocytes reside on peptides of Mr approximately equal to 62,000-64,000. In this study, a two-dimensional gel electrophoresis method for peptide mapping was used to investigate and compare the structure of beta 1 - and beta 2-adrenergic receptor subtypes. When the photoaffinity labeled Mr approximately equal to 62,000 peptides from the beta 2-adrenergic receptors of rat lung and erythrocyte are subjected to simultaneous proteolysis using Staphylococcus aureus V8 proteinase or papain, exactly the same peptide fragments are generated from each subunit. In contrast, when the Mr approximately equal to 62,000 peptide containing the beta 1-adrenergic receptor-binding subunit derived from the rat heart is proteolyzed simultaneously with the Mr approximately equal to 62,000 peptide containing the beta 2-adrenergic receptors from either lung or erythrocyte, the peptide fragments generated are distinctly different. Peptide maps of beta 1-adrenergic receptors from the myocardial tissue of different species (pig versus rat) yield slightly different maps while the maps derived from the beta 2-adrenergic receptors of hamster lung and rat lung or erythrocytes reveal no interspecies differences. These data suggest: 1) alterations in the primary structure of the beta-adrenergic receptor may be responsible for the pharmacological specificities characteristic of beta 1- and beta 2-adrenergic receptor subtypes; and 2) alterations in the primary structure of similar beta-adrenergic receptor subtypes across different species may relate to the magnitude of their phylogenetic differences.

Human T lymphocyte proliferation induced by a pan-T monoclonal antibody (anti-Leu 4): heterogeneity of response is a function of monocytes.

Anti-Leu-4 is a murine monoclonal antibody that defines a molecule of 20,000 to 25,000 daltons present on all mature T lymphocytes in man. When cultured in the presence of 10 to 1000 ng/ml anti-Leu-4, the T cells of most individuals proliferate with peak responses on the third day of culture. T cells of both helper and suppressor lineages proliferate, but only in the presence of monocytes. Approximately 40% of individuals tested responded weakly or not at all to anti-Leu-4, despite normal responses to other stimuli. The variation in responsiveness between individuals could not be explained by differences in Leu-4 antigen density on the surface of T cells, differences in the rate of Leu-4 antigen modulation, or structural differences in the Leu-4 molecule as defined by the method of two-dimensional polyacrylamide gel electrophoresis. In the presence of monocytes from high responders, the T cells from low responders proliferated vigorously to anti-Leu-4, whereas monocytes from low responders failed to support proliferation by high responder T cells. On the other hand, low responder monocytes did not prevent T cells from proliferating in the presence of high responder monocytes. These results suggest that the failure of some individuals to respond to anti-Leu-4 is due to the absence or dysfunction of an essential monocyte population.

An efficient two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis method for simultaneous peptide mapping of protein contained in a mixture

A method for simultaneous peptide mapping of polypeptides contained in a mixture is presented. The polypeptides were first separated by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The strip of gel containing these unstained polypeptide bands was subsequently embedded perpendicular to the direction of electrophoresis in the stacking gel of a second gel. The proteolytic enzymes, loaded on top of the second gel, were brought in contact with the substrates through moving boundary electrophoresis. The peptides thus generated were then resolved by electrophoresis in a gradient gel. a polychromatic silverstaining method added an extra dimension to the identification and characterization of the peptides in the maps obtained in that specific peptides got specific colors. Moreover, the sensitivity of this method was illustrated by the demonstration that original quantities in the submicrogram range of nonradioactive proteins (exemplified here by the structural proteins of densonucleosis virus) largely sufficed for satisfactory maps. Other advantages of this procedure over current methods included (i) the elimination of the purification step (and consequently virtually no loss or contamination), (ii) that only the strict minimum of material (necessary for the ultimate visualization of the maps) had to be used, (iii) that no special two-dimensional electrophoresis equipment was needed, and (iv) the consistency, speed, and simplicity of the method.

Two-dimensional gel electrophoresis method for investigation of human plasma proteins: detection of subtle changes during filtration leukapheresis.

Special problems are associated with analysis of human plasma proteins by standard "high-resolution" two-dimensional gel electrophoresis methods in which isoelectric focusing is followed by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels (SDS-PAGE). Individual plasma proteins are often separated into overlapping groups of multiple spots, and identification of individual spots is further confounded by genetic variation. Analytical recovery of components of high molecular mass is also low or variable. These problems may be reduced or overcome by use of a "low-resolution" method consisting of electrophoresis of native proteins at pH 8.6 in an agarose gel followed by SDS-PAGE without added reducing agent. About 60 proteins can be resolved, most as single spots. About 25 of these proteins have been "mapped," and tentatively identified. We have examined 119 plasma samples taken from six donors who were undergoing filtration leukapheresis and 10 donors who were undergoing centrifugation leukapheresis or plateletpheresis. In all cases, passage of blood through a nylon filter induced a significant increase in a doublet of spots tentatively identified as complement component C3c. This was detected in the effluent from the filter throughout the first 30 min of filtration, and to a lesser extent in the venous blood. These spots were not induced by the centrifugation procedures. One filtration donor also showed increased acute-phase proteins 24 h after the procedure.

Evidence for two dissimilar polypeptide chains in the beta 2 subunit of hexosaminidase.

The major isoenzymes of human hexosaminidase have the structures alpha beta 2 (hex A) and 2 beta 2 (hex B). In this study, we present evidence that the beta 2 subunit of hex B and hex BA (the form of hex B derived from hex A) is composed of two nonidentical polypeptide chains. We have called these chains beta a and beta b. They have similar molecular weights (25,000) but have pI values that differ by 1 unit. We have used a two-dimensional analytical gel electrophoresis method in combination with peptide mapping to compare the primary sequence structure of the two beta chains. In this method, the polypeptide chains of hex B or hex BA were first separated by isoelectric focusing in 8.5 M urea. The separated chains were subjected to partial proleolytic digestion in the stacking gel of a second NaDodSO4/polyacrylamide gel with subsequent separation of peptides by electrophoresis into the second gel. Partial digestion by protease V8 or papain showed that the beta a and beta b species have distinct primary structures, neither of which was similar to that of the alpha chain. On the basis of these results, we suggest that the beta 2 subunit of hexosaminidase has the structure of beta a beta b. The possibility that the distinct beta chains are encoded by a single gene is discussed in the light of genetic and other data.

Human Skin Cells Synthesize HLA-DR Molecules of the Same Charge and Molecular Weight as those Synthesized by Autologous Lymphocytes

Between 5 and 14% of human skin cells, separated by trypsinization, express HLA-DR antigen as detected by indirect immunofluorescence with monoclonal anti-DR antibody. To determine the source and structure of these DR molecules, skin cell suspensions were biosynthetically labeled with 35S-methionine and the radiolabeled DR molecules were analyzed by the method of two-dimensional polyacrylamide gel electrophoresis. The results indicated the presence of heavy, intermediate, and light chains indistinguishable from those of B lymphocyte DR. Gels in which DR immunoprecipitates from skin cells and autologous lymphocytes were run simultaneously confirmed this finding. Thus, skin cells and lymphocytes synthesize HLA-DR antigen of identical charge and molecular weight.

Cultured Human Epidermal Cells Do Not Synthesize HLA-DR

All nucleated cells express HLA-A, B, and C antigens. However, only a few cells, including epidermal cells, demonstrate HLA-DR antigens which are potent transplantation immunogens in man. The current study was undertaken to determine if epidermal cell continue to synthesize and/or express HLA-DR antigens after prolonged in vitro culture. Epidermal cells cultured for 7 days or more no longer stimulated allogeneic lymphocytes in the epidermal cell-lymphocyte reaction. Indirect immunofluorescence light microscopy of cultured cells using mouse monoclonal antibody to HLA-DR antigen confirmed that these cells do not express HLA-DR antigens whereas they retain beta 2-microglobulin. Detergent extracts of 12-day cultured epidermal cells biosynthetically labeled with 35 S-methionine were immunoprecipitated with monoclonal anti-DR antibody and analyzed by the method of two-dimensional polyacrylamide gel electrophoresis. No radiolabeled proteins were found on these gels in the regions where HLA-DR molecules are known to migrate. These data indicate that HLA-DR antigen is absent from cultured epidermal cells. Finally, we describe a technique for growing epidermal cells on a gelatin membrane which allows subsequent removal of intact cell monolayers from the culture dish. Such monolayers may be useful for purposes of transplantation.

Two-dimensional gel electrophoresis of chicken skeletal muscle proteins with agarose gels in the first dimension

An improved method of two-dimensional gel electrophoresis is described. The method is specifically developed for preparing a “protein map” of chicken skeletal muscle, and is found to be applicable to the analysis of most protein constituents including high molecular ones, such as myosin heavy chain, without using any detergents in the first dimension. Omission of detergents from the focusing medium results in two advantages. (i) The first-dimension isoelectric focusing pattern can be recorded by taking a photograph of the gel prior to the second-dimension electrophoresis, so that even a close doublet band in the first dimension, which forms one spot in the second dimension, can be found heterogeneous in component by examining the first-dimension pattern of the same gel. (ii) Since peptides of relatively large molecular weights can be analyzed by first-dimension isoelectric focusing, complex formation between polypeptides with different isoelectric points is demonstrable. For example, troponin T, troponin I, and troponin C are found by two-dimensional gel electrophoresis to form a complex in a 4 m urea solution, and so are troponin I and troponin C in a 5 m urea solution.

Application of on-line two-dimensional liquid chromatography in comparative proteome analysis of antlers with different growing stages

A method of on-line two-dimensional weak anion exchange chromatography-reversed-phase liquid chromatography (2D WAX-RPLC) was established, and applied for the comparative proteome study of antlers with four different growing stages. A five-protein-mixture was used as the sample to evaluate the reproducibility of the system. In addition, the capacity of such a system for the relative quantitative analysis was demonstrated by analyzing protein mixtures with different concentration ratios. Furthermore, five different lysis buffers were used to extract proteins from antlers with different growing stages. After the protein separation with 2D WAX-RPLC, the fractions with maximum peak height ratio larger than 2 were collected, digested, and further identified by micro-reversed-phase liquid chromatography-tandem mass spectrometry (microRPLC-MS/MS). In total, 22 differential proteins were identified from 9 fractions. Our experimental results demonstrated that 2D WAX-RPLC based protein separation has the advantages of good reproducibility and automation, and might be a complementary method of two-dimensional gel electrophoresis for comparative proteome study.

69] Purification of human leukocyte interferon by two-dimensional polyacrylamide gel electrophoresis

The homogeneous human leukocyte interferon can be obtained by devising a purification scheme that includes the elimination of heterogeneity by chemical oxidation with sodium periodate, molecular sieve column chromatography, affinity column chromatography, and a modification of the two-dimensional gel electrophoresis technique. A modified two-dimensional gel electrophoresis system is used to further purify the human leukocyte interferon preparations. The modifications made are (1) the omission of reducing agents, such as β-mercaptoethanol, in all solutions and (2), the increased length of the isoelectric focusing gel from l I cm to 24 cm with the corresponding increase of the second-dimension sodium dodecyl sulfate (SDS) gel slab to 35 cm wide by 30 cm in height. Homogeneous spots of two forms of human leukocyte interferons are obtained with this method, each with a final specific activity of about 10 9 units per milligram of protein. This method of two-dimensional gel electrophoresis of periodatetreated preparations can be used to isolate two distinct species of human leukocyte interferon molecules differing in human to bovine activity ratios.

Surface labeling of membrane glycoproteins and their drastic changes during development of Dictyostelium discoideum.

Surface glycoproteins on plasma membranes from Dictyostelium discoideum were labeled by sodium metaperiodate oxidation and sodium boro[3H]hydride reduction. The amount of incorporation of tritium from NaB3H4 reached a plateau after 10 min at a periodate concentration of 20 mM. The density analysis carried out by sucrose density-gradient centrifugation showed that the plasma membranes were selectively labeled by this technique. About 84% of the radioactivity incorporated was released by hydrolysis with 0.25 M H2SO4 for 3 h at 100 degrees C. The released materials were eluted at a bed volume after chromatography using a Sephadex G-50 column. From the paper chromatographic analysis of the eluate, four radioactive spots were detected. Two of them were glycerol and glyceraldehyde and the other two spots seemed to be oligosaccharides. Using the above method, the plasma membranes from aggregation-phase cells were labeled four times more than those from growth-phase cells. The labeled plasma membrane fraction during the aggregation phase was separated into at least 39 distinct glycoprotein bands on a one-dimensional dodecylsulfate/polyacrylamide gel. Six of the 39 bands increased markedly in density and two new bands appeared. In contrast, four bands decreased in density in the aggregation phase. Using this method, the surface glycoproteins can be analyzed directly be gel electrophoresis of the lysate of labeled whole cells without preparing the plasma membranes. Changes during development of glycoproteins on outer cell surfaces were also confirmed by O'Farrell's method of two-dimensional polyacrylamide gel electrophoresis. Utilizing this technique, glycoproteins on plasma membranes of D. discoideum were separated into 63 individual spots; 45 of these 63 spots changed during the early developmental course of D. discoideum. This is in contrast to the slight change in the soluble and membrane proteins during this phase. This fact also suggests that the glycoproteins have important roles in the process of aggregation.

Protein identifications of O'Farrell two-dimensional gels: locations of 81 Escherichia coli proteins.

The two-dimensional gel electrophoresis method of P. H. O'Farrell readily resolves approximately 1,000 proteins from whole-cell homogenates. We have found that the location of most individual proteins is sufficiently reproducible and precise to permit different laboratories to exchange information about them. We present the location of 81 Escherichia coli structural proteins, binding proteins, enzymes, and factors, identified with the aid of purified proteins supplied to us by many investigators.