Showing papers on "Gel electrophoresis published in 1987"
TL;DR: A discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system for the separation of proteins in the range from 1 to 100 kDa is described, and the omission of glycine and urea prevents disturbances which might occur in the course of subsequent amino acid sequencing.
11,290 citations
TL;DR: Small amounts of myoglobin, beta-lactoglobulin, and other proteins and peptides can be spotted or electroblotted onto polyvinylidene difluoride membranes, stained with Coomassie Blue, and sequenced directly, suggesting that PVDF membranes are superior supports for sequence analysis of picomole quantities of proteins purified by gel electrophoresis.
4,869 citations
TL;DR: The development of two new methods for detecting and localizing single base changes in cloned and genomic DNA are described, likely that the use of both methods will be complementary, resulting in the detection of a very large fraction of all possible substitutions.
Abstract: Publisher Summary This chapter discusses the detection and localization of single base changes by denaturing gradient gel electrophoresis For most purposes, single-stranded deoxyribonucleic acid (DNA) probes 140–1000 bases in length are used, which allows about half of this number of base pairs to be screened for base changes in a single gel lane The chapter discusses the use of single-stranded RNA probes It describes the development of two new methods for detecting and localizing single base changes in cloned and genomic DNA In both procedures, a single-stranded, radioactively labeled probe of wild-type sequence is annealed to cloned or genomic DNA If the DNA under test carries a single base change, a hybrid double-stranded species containing a mismatch is formed In the first procedure, either a ribonucleic acid (RNA) or a DNA probe is used, and the mismatched duplex is separated from the perfectly paired, wild type duplex by electrophoresis in a denaturing gradient gel In the second procedure, the probe is composed of single-stranded RNA and the mismatch is cleaved by ribonuclease; the resulting cleaved products are examined by polyacrylamide gel electrophoresis and autoradiography It is estimated that each method will detect approximately 50% of all possible substitutions, insertions, or deletions in a probed region Although there is some overlap in the base changes that are detected with the two procedures, it is likely that the use of both methods will be complementary, resulting in the detection of a very large fraction of all possible substitutions and they should be particularly useful in mapping and diagnosing small mutations that result in genetic disease This method has led to the detection of neutral polymorphisms for genetic linkage studies The chapter lists all the equipment needed to prepare and run the gels and also describes the hybridization procedure for RNA probes
938 citations
TL;DR: To obtain direct evidence that ubiquitin is a component of PHF, PHF were treated with concentrated formic acid and digested with lysylendopeptidase; ubiquitIn-derived peptides were then identified by reversed-phase high-performance liquid chromatography.
Abstract: Paired helical filaments (PHF), which constitute a distinct type of pathological neuronal fiber, are the principal constituent of neurofibrillary tangles that occur in the brain of patients with Alzheimer's disease. Their insolubility in sodium dodecyl sulfate and urea has prevented the analysis of their subunit composition by gel electrophoresis. A monoclonal antibody (DF2) was isolated that specifically labeled PHF at both the light and electron microscopic levels. It labeled a small polypeptide (5 kilodaltons) that was shown to be ubiquitin in immunoblots of the soluble fraction of brain homogenates. To obtain direct evidence that ubiquitin is a component of PHF, PHF were treated with concentrated formic acid and digested with lysylendopeptidase; ubiquitin-derived peptides were then identified by reversed-phase high-performance liquid chromatography. Two fragments in the PHF digest were identified as derived from ubiquitin by protein sequencing. This procedure should make possible definitive identification of other PHF components.
800 citations
TL;DR: The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate oligonucleotide probes for molecular cloning and/or used to search sequence data bases for related proteins.
Abstract: We have developed a general two-step method for obtaining peptide fragments for sequence analysis from picomole quantities of proteins separated by gel electrophoresis. After separation by one- or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out, and each protein is digested in situ by proteolytic enzymes such as trypsin or staphylococcal V-8 protease. The resulting peptide fragments are separated by narrow-bore reverse-phase HPLC, collected, and sequenced in a gas-phase sequenator. Excellent peptide recoveries and the absence of extraneous contaminants in the separation of the peptide fragment mixture allow the generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate oligonucleotide probes for molecular cloning and/or used to search sequence data bases for related proteins. This method has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from one- and two-dimensional polyacrylamide gels.
765 citations
TL;DR: Calculation of the theoretical pattern of thermal stability of DNA molecules of known sequence, together with calculation of the expected changes in electrophoretic mobility in gels under denaturing conditions, is the first useful step toward searching for sequence changes by means of denatured gradient gels and in designing a gradient gel for the preparative isolation of mutants after intensive mutagenesis.
Abstract: Publisher Summary This chapter discusses the application of computational simulation of deoxyribonucleic acid (DNA) melting for denaturing gradient gel electrophoresis Calculation of the theoretical pattern of thermal stability of DNA molecules of known sequence, together with calculation of the expected changes in electrophoretic mobility in gels under denaturing conditions, is the first useful step toward searching for sequence changes by means of denaturing gradient gels and in designing a gradient gel for the preparative isolation of mutants after intensive mutagenesis The calculations permit a reasonably reliable test of the experimental prospects for new hypotheses and experimental designs They show, for example, that about 50–70% of all possible single base changes that might occur within the human β-globin gene cluster would be detectable using the denaturing gradient system after the restriction fragments of the genome are hybridized with labeled, single strands corresponding to the same fragments of the normal sequence The calculations are easy to execute for any sequence on a digital computer They provide an indication of the regions within the sequence where base changes are likely to be detected, the magnitude of the effects that can be expected, and guidance as to the choice of restriction sites for fragmentation The theoretical calculation is sufficiently reliable to serve as a means for the evaluation of some thermodynamic parameters relevant to melting from gel data and to signal anomalous properties The chapter discusses theoretical treatment of the helix-random chain transition for complex sequences, the melting map program MELT, the mobility program MU, and so on The melting calculation is useful in several different applications The most elementary result is a description of the probability that each base is helical (or nonhelical) at specified temperatures
512 citations
TL;DR: The correlation of dephosphorylation of p28 with the inhibition of protein synthesis and the relatively low abundance of the eIF-4F complex suggest that eIF -4F plays a role in the translational control of mRNA binding.
486 citations
TL;DR: Results suggest that the two polypeptides are in close association and are part of the cytochrome b, provide explanation for the molecular weight discrepancies previously reported for the protein, and further support the involvement of the Cytochrome in superoxide production in human neutrophils.
Abstract: A new method has been developed for purification of cytochrome b from stimulated human granulocytes offering the advantage of high yields from practical quantities of whole blood. Polymorphonuclear leukocytes were treated with diisopropylfluorophosphate, degranulated and disrupted by nitrogen cavitation. Membranes enriched in cytochrome b were prepared by differential centrifugation. Complete solubilization of the cytochrome from the membranes was achieved in octylglucoside after a 1-M salt wash. Wheat germ agglutinin-conjugated Sepharose 4B specifically bound the solubilized cytochrome b and afforded a threefold purification. Eluate from the immobilized wheat germ agglutinin was further enriched by chromatography on immobilized heparin. The final 260-fold purification of the b-type cytochrome with a 20-30% yield was achieved by velocity sedimentation in sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified preparation revealed two polypeptides of Mr 91,000 and Mr 22,000. Treatment of the 125I-labeled, purified preparation with peptide:N-glycosidase F, which removes N-linked sugars, decreased relative molecular weight of the larger species to approximately 50,000, whereas beta-elimination, which removes O-linked sugars, had little or no effect on the mobility of the Mr-91,000 polypeptide. Neither of the deglycosylation conditions had any effect on electrophoretic mobility of the Mr-22,000 polypeptide. Disuccinimidyl suberate cross-linked the two polypeptides to a new Mr of 120,000-135,000 by SDS-PAGE. Antibody raised to the purified preparation immunoprecipitated spectral activity and, on Western blots, bound to the Mr-22,000 polypeptide but not the Mr-91,000 polypeptide. Western blot analysis of granulocytes from patients with X-linked chronic granulomatous disease revealed a complete absence of the Mr-22,000 polypeptide. These results (a) suggest that the two polypeptides are in close association and are part of the cytochrome b, (b) provide explanation for the molecular weight discrepancies previously reported for the protein, and (c) further support the involvement of the cytochrome in superoxide production in human neutrophils.
435 citations
TL;DR: Plasma GSHPx, like the erythrocyte enzyme, was found to contain approximately four atoms of selenium per mole of protein and is a glycoprotein, and radioimmunoassay showed that less than 0.13% of the antigenically detectable protein is found in the purified GSHpx from plasma.
399 citations
TL;DR: A phosphorylated glycoprotein was purified from the mixture of proteins extracted by demineralization of rat bone with 0.5 M EDTA in 4 M guanidinium chloride and showed a high level of purity.
360 citations
TL;DR: High-performance capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been developed for the separation and molecular weight (MW) determination of peptides and proteins and the expected common intercept of log mobility vs. mobility for the SDS protein complexes was observed.
TL;DR: The chapter mentions the development of three new techniques that allow the fractionation and analysis of DNA molecules on a size scale much larger than previously possible, and describes the new techniques and demonstrates examples of their applicability for the analysis of bacterial genomes and unicellular eukaryotic genomes.
Abstract: Publisher Summary This chapter discusses the processes related to the purification, specific fragmentation, and separation of large deoxyribonucleic acid (DNA) molecules. The chapter mentions the development of three new techniques that allow the fractionation and analysis of DNA molecules on a size scale much larger than previously possible. The current methods permit the routine handling of DNAs up to 1.5 million base pairs (bp). The first technique involves the preparation of unbroken genomic DNAs inside agarose gels. The second involves the digestion of such DNAs in agarose with restriction nucleases that produce discrete, large fragments. The third technique, pulsed-field gel (PFG) electrophoresis, allows the size separation of DNAs ranging from 10,000 bp (10 kb) to more than 1.5 million bp (1.5 Mb). The chapter describes the new techniques and demonstrates examples of their applicability for the analysis of bacterial genomes and unicellular eukaryotic genomes. The chapter describes the procedure for preparing intact DNA. Ordinary DNA preparative procedures are carried out in solution. In a typical DNA preparation, the walls of the cells are first removed by appropriate enzymatic treatment. The resulting spheroblasts or protoplasts are then broken open by the destruction of their cell membranes with detergents and a metal chelator. This produces a complex mixture of DNA, ribonucleic acid (RNA), and proteins. Treatment of the mixture with proteases and RNases may remove some of the unwanted components. Additional proteins are removed by chemical extraction of the DNA solution with phenol, and the DNA is concentrated by alcohol precipitation and centrifugation. To obtain clean DNA preparations that do not have contaminants, which interfere with subsequent analytical or preparatory procedures, it is frequently necessary to repeat some of these steps. It is also essential that, during preparation, DNAs are not exposed unnecessarily to DNases. The chapter also discusses the pulsed-field gel electrophoretic separation of intact chromosomal DNA molecules, the specific fragmentation of high-molecular-weight DNA, and the applications of large DNA technology for the study of organisms with simple genomes.
TL;DR: The structural and functional characteristics of the platelet-derived endothelial cell growth factor are distinct from previously characterized endothelium cell mitogens with affinities for heparin.
TL;DR: It is reported here that Cytochrome b−245 has two subunits, a 23K protein and the previously described 76–92K glycoprotein, which remain associated with the haem of the cytochrome through affinity and gel filtration chromatography and sucrose gradient centrifugation, and exhibit a similar distribution in a pH gradient.
Abstract: Phagocytosis by neutrophils and other 'professional' phagocytic cells is accompanied by a microbicidal burst of non-mitochondrial respiration. Cytochrome b-245 is the only clearly defined component of this oxidase system and its absence provides the molecular basis of X-linked chronic granulomatous disease (CGD), in which a profound predisposition to infection results from complete failure of this respiratory burst. Purification of the cytochrome has proved difficult, with uniform disagreement regarding the identity of its apoprotein, descriptions of its relative molecular mass (Mr) on SDS-polyacrylamide gel electrophoresis (PAGE) ranging from 10,000 to 127,000 (10-127K). I report here that it has two subunits, a 23K protein and the previously described 76-92K glycoprotein. These subunits are closely linked and remain associated with the haem of the cytochrome through affinity and gel filtration chromatography and sucrose gradient centrifugation, and exhibit a similar distribution in a pH gradient. Neither protein was detected in the cells of five patients with X-linked CGD whereas both were present in two with the form of this disease with autosomal recessive inheritance.
TL;DR: Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes and displays catalytic characteristics similar to the enzyme in intact microsome in the aromatization of androstenedione, 19-hydroxyandrosthenione and 19-oxoandrostenedion.
TL;DR: DNA sequence analysis indicates that the four classes of cDNA were derived from transcripts of the alpha 1, zeta, beta h1 and epsilon globin genes, which correspond to globins of the newly established blood system.
Abstract: The pattern of protein synthesis in 8-, 9- and 10-day post coitum (p.c.) mouse embryos was examined by 2-D gel electrophoresis of [35S]methionine-labelled proteins. Of the 600–800 polypeptides detected only one, a 14 X 10(3) Mr (14K) protein, was found to accumulate over this period. To isolate cDNA clones that potentially encode this protein, 32P-labelled cDNA was synthesized from 9 and 10 days p.c. embryo poly(A) +RNA, and used for the differential screening of an 8.5-day p.c. mouse embryo cDNA library cloned in lambda gt10. Six clones that hybridized strongly to the 10-day probe were purified and their inserts subcloned into plasmid vectors. Cross hybridization and restriction mapping of these inserts indicate that they fall into four distinct groups. Each of these hybridize with transcripts of approximately 600 nucleotides, which accumulate in the embryo from 9 to 10 days p.c. Expression was barely detectable in adult tissues and restricted to liver and spleen. Expression of one of these clones, 10.1, was examined by in situ hybridization of 35S-labelled RNA probes to 8.5-12.5 day p.c. embryo sections. Strong hybridization was observed in yolk sac blood islands, fetal liver and embryonic erythrocytes, suggesting that 10.1A encodes an erythrocyte-specific protein. DNA sequence analysis indicates that the four classes of cDNA were derived from transcripts of the alpha 1, zeta, beta h1 and epsilon globin genes. Labelling of 10-day p.c. erythrocyte proteins with [35S]methionine, followed by 2-D gel electrophoresis, clearly demonstrates that the most abundant polypeptide migrates to the same position as the 14K protein which accumulates from 8 to 10 days p.c. Thus the only abundant transcripts and corresponding proteins that change over a period of profound morphogenetic change correspond to globins of the newly established blood system.
TL;DR: This chapter describes a simple method that permits the expression of cloned sequences in Escherichia coli as well as in cell-free in vitro systems of eukaryotic or prokaryotic origin, using a single expression unit using a T5 promoter-based transcription–translation system.
Abstract: Publisher Summary This chapter discusses a T5 promoter-based transcription–translation system for analyzing proteins in vitro and in vivo . The chapter describes a simple method that permits the expression of cloned sequences in Escherichia coli as well as in cell-free in vitro systems of eukaryotic (wheat germ, reticulocyte, and HeLa cells) or prokaryotic ( E. coli ) origin, using a single expression unit. The expression of cloned genes in heterologous systems in vitro and in vivo has been instrumental in the identification and analysis of gene products and their derivatives. The essential element of the unit is a promoter derived from coliphage T5 that is utilized by E. coli ribonucleic acid (RNA) polymerase. It efficiently directs the synthesis of capped or uncapped mitochondrial RNA (mRNA) in vitro . Translation of such RNAs in the presence of [ 35 S] methionine yields single proteins of such high specific activity and purity that they can be directly analyzed by gel electrophoresis without the necessity of prior immunoprecipitation. The chapter also describes the application of the method for the study of structure/function relationships of proteins, including (1) protein synthesis in vivo and in vitro , (2) the translocation of proteins into and through membranes, and (3) the interruption of translation at predetermined sites for the generation and characterization of truncated proteins. Plasmid pDS5 and its derivatives 5 are members of a plasmid family developed for the study of transcriptional signals. The chapter also summarizes the essential properties of the pDS5 system.
TL;DR: Excellent resolution of chromosomal DNA molecules from Saccharomyces cerevisiae, Candida albicans and Schizosaccharomycles pombe has been obtained using alternating contour-clamped homogeneous electric field (CHEF) gel electrophoresis.
Abstract: Excellent resolution of chromosomal DNA molecules from Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe has been obtained using alternating contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The largest of these molecules is greater than 5 Mb in size and is resolved after 130 hours in a 0.6% agarose gel at a field strength of 1.3 V/cm and a switching interval of 1 hour. Separation of concatamers of phage lambda DNA reveals four regions of resolution in alternating CHEF gel electrophoresis. There are two regions of good resolution in which mobility approximates a linear function of molecular weight. These are separated by a region of lower resolution and bounded at high molecular weights by a region of little or no resolution. The four regions are of practical and possibly theoretical importance.
TL;DR: Comparison of the purified topoisomerase II from amsacrine-resistant P388 with that from amSacrine-sensitive P388 demonstrated that each cell type contained both p180 and p170; however, the relative amounts of the two proteins were consistently different in the two cell types.
TL;DR: An SDS-PAGE technique is described that allows identification of the antimicrobial activity of a peptide secreted by a strain ofediococcus acidilactici that has an antimicrobial property against several baeteria associated with food.
Abstract: An SDS-PAGE technique is described that allows identification of the antimicrobial activity of a peptide secreted by a strain ofPediococcus acidilactici. This peptide has an antimicrobial property against several baeteria associated with food. This technique enables detection of the specific peptide (or protein) band(s) associated with the inhibitory effect which can then be eluted from the gel for further studies.
TL;DR: The neuron-binding and neurite outgrowth-promoting properties of p30 as well as the developmental regulation of its content in brain tissue suggest a role in neuronal growth.
TL;DR: It is demonstrated that all three proteins are related isoforms, with two of the isoforms containing phosphate, and a relatively simple and rapid purification of the proteins employing both ion-exchange and gel filtration chromatography.
TL;DR: It could be shown that by this method dsRNA molecules may be differentiated which differ only in one base-pair, or proteins differing in one amino acid only, as a particular advantage, temperature-gradient gel electrophoresis allows the study of conformational transitions of biopolymers which have not been purified.
TL;DR: This cell line may be a useful model for investigating the metabolism of newly formed HDL, and Immunoblotting after gradient gel electrophoresis of concentrated medium revealed that a significant fraction of apoA-I in the uncentrifuged medium was in a lipid-poor or lipid-free form.
TL;DR: The high-resolution capacity of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) makes it an excellent tool for the analysis and characterisation of complex protein mixtures and some of the biomedical applications to screening of body fluids, genetic diseases, inborn errors of metabolism, cancer and neoplastic transformation are discussed.
TL;DR: Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing and recognized not only native but also partially deglycosylated protein.
Abstract: Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing. Results of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated the approximate molecular weight of the native protein to be 85-100-KDa. On the basis of a molecular weight of 85-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 1.2 X 10(4) M-1 cm-1. The isoelectric point of the enzyme, as deduced by chromatofocusing, was about 4.5. The purified enzyme is remarkably stable at 0 degree C. Thermal inactivation studies have shown that the enzyme retained 40% of its activity after being subjected to 68 degrees C for 10 minutes, and the enzyme exhibited a broad temperature optimum with maximum catalytic activity at 58 degrees C. The Km of the enzyme for phytate and p-nitrophenylphosphate is about 40 uM and 265 uM, respectively, with an estimated turnover number of the enzyme for phytate of 220 per sec. Enzymatic deglycosylation of phytase by Endoglycosidase H lowered the molecular weight of native enzyme from 85-100-KDa to about 76-KDa; the digested phytase still retained some carbohydrate as judged by positive periodic acid-Schiff reagent staining of the electrophoresed protein. Immunoblotting of the phytase with monoclonal antibody 7H10 raised against purified native enzyme recognized not only native but also partially deglycosylated protein.
TL;DR: Results demonstrate that the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel from rabbit skeletal muscle contains two distinct high molecular weight subunits of 175,000 and 170,000.
TL;DR: Results indicate that the placental anticoagulant protein (PAP), purified from the soluble fraction of human placenta, inhibits coagulation by binding to phospholipid vesicles.
Abstract: An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and Mono S (Pharmacia). The yield of the purified protein was approximately 20 mg from one placenta. The purified protein gave a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein prolonged the clotting time of normal plasma when clotting was induced either by brain thromboplastin or by kaolin in the presence of cephalin and Ca2+. It also prolonged the factor Xa induced clotting time of platelet-rich plasma but did not affect thrombin-induced conversion of fibrinogen to fibrin. The purified placental protein completely inhibited the prothrombin activation by reconstituted prothrombinase, a complex of factor Xa-factor Va-phospholipid-Ca2+. The placenta inhibitor had no effect on prothrombin activation when phospholipid was omitted from the above reaction. Also, it neither inhibited the amidolytic activity of factor Xa, nor did it bind to factor Xa. The placenta inhibitor, however, did bind specifically to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that the placental anticoagulant protein (PAP) inhibits coagulation by binding to phospholipid vesicles. The amino acid sequences of three cyanogen bromide fragments of PAP aligned with those of two distinct regions of lipocortin I and II with a high degree of homology, showing that PAP is a member of the lipocortin family.
TL;DR: Human class II histocompatibility antigens were purified from the Epstein-Barr virus-transformed human B lymphoblastoid cell line LG-2 by immunoaffinity chromatography for the first time all three subsets have been prepared as nonradioactive materials on a milligram scale.
TL;DR: The individual antigenic components present in microgram quantities of complex mixtures can be separated reproducibly by polyacrylamide gel electrophoresis and transferred onto nitrocellulose blots and can induce maximal lymphoproliferative responses in 30-60 microtitre wells.