Isoelectrical focusing of connectin by agarose gel electrophoresis

Two-dimensional gel electrophoresis using 0.5% agarose gel in the 1st dimension and 2-12% gradient polyacrylamide gel in the 2nd dimension succeeded in the isoelectrical focusing of connectin, a giant myofibrillar protein of approximately 3,000 kDa. Immunoblotting with an anti-connectin monoclonal antibody, SM1-36-2, following the two-dimensional gel electrophoresis, demonstrated that connectin was an acidic protein with an estimated pI of approximately 5.7.     

Levels of ribosomal protein S1 and elongation factor G in the growth cycle of Escherichia coli.

The relative levels of ribosomes, ribosomal protein S1, and elongation factor G in the growth cycle of Escherichia coli were examined with two-dimensional polyacrylamide gel electrophoresis. Nonequilibrium pH gradient polyacrylamide gel electrophoresis was used in the first dimension, and polyacrylamide gradient-sodium dodecyl sulfate gel electrophoresis was used in the second dimension. The identities of protein spots containing S1 and elongation factor G were confirmed by radioiodination of the proteins and peptide mapping of the radiolabeled peptides. The levels of ribosomes and ribosomal protein S1 were coordinately reduced during transition from exponential phase to stationary phase. There was no accumulation of S1 in the stationary phase. In marked contrast, the level of elongation factor G showed no significant change from exponential phase to stationary phase. The relative level of elongation factor G compared with ribosomes or S1 increased by about 2.5-fold during transition from exponential phase to stationary phase. The results show that there are differences between the regulation of the levels of elongation factor G and of ribosomal proteins, including S1, apparent during the transition from exponential to stationary phase.     

Two-dimensional gel electrophoresis to separate large RNA molecules such as messenger RNAs

This paper describes a two-dimensional gel electrophoresis method for separating large RNA molecules such as messenger RNAs. RNAs were at first separated on a polyacrylamide plus agarose composite gel and subjected to a second dimension electrophoresis on a polyacrylamide gel containing urea. This method is illustrated by analyses of poly(A)+ yeast RNAs. About 80 discrete spots were detected on the gel, when RNAs from 1000 to 3500 nucleotides in size were examined.     

An Examination of Corynebacterium spp. by Gel Electrophoresis

S ummary . The cell free extracts of 24 organisms of the genus Corynebacterium were examined by starch gel electrophoresis for esterase, catalase and peroxidase activity. The protein components of eleven of these extracts were also detected after electrophoresis in polyacrylamide gel. The results indicate a separation of the members of the genus into distinct subgroups comparable to those obtained by grouping strains according to habitat.     

Effect of different carbon sources and cold shock on protein synthesis by a psychrotrophic Acinetobacter sp

The induction of proteins after a 25 to 5 degrees C cold shock in the psychrotrophic Acinetobacter HH1-l was examined using two-dimensional polyacrylamide gel electrophoresis. In addition, effects of various carbon sources (acetate, Tween 80, and olive oil) on protein synthesis after cold shock were assessed. HH1-1 responded to cold shock by synthesizing both cold shock proteins (csps) and cold acclimation proteins (caps). The synthesis of two csps (89 and 18) was increased 2 h after cold shock by the cells, regardless of the carbon source provided. An additional csp (csp 12), with an estimated molecular mass of 12 kDa, was observed in cells grown in olive oil only. Csp 12 was also synthesized when cells were incubated at 30 degrees C, suggesting that this protein may serve as a general stress protein. In addition to csps, caps were observed post cold shock at 72 h in acetate-grown cells and at 140 h in cells grown in Tween 80 and olive oil. Induction of cold-acclimated periplasmic proteins was observed for cells grown in olive oil only, suggesting cells grown in olive oil may be stressed by low temperatures to a greater extent than cells grown in either acetate or Tween 80.     

Two-dimensional gel electrophoresis of the membrane-bound protein complexes, including photosystem I, of thylakoid membranes in the presence of sodium oligooxyethylene alkyl ether sulfate/dimethyl dodecylamine oxide and sodium dodecyl sulfate

Two-dimensional polyacrylamide gel electrophoresis (PAGE), using a mixture of sodium oligooxyethylene alkyl ether sulfate and dimethyl dodecylamine oxide as detergents (AES-DDAO mixture) in the first dimension and sodium dodecyl sulfate (SDS) in the second dimension, was developed and applied to an analysis of the photosystem I (PS I) complex in thylakoid membranes prepared from spinach chloroplasts. When thylakoid membranes of chloroplasts were solubilized directly in the AES-DDAO mixture and subjected to PAGE in the presence of these detergents as the first dimension, some protein complexes containing chlorophyll were observed. The protein components in these complexes separated into an array of polypeptide spots when the strip of gel after PAGE in the first dimension was subjected to PAGE in the presence of SDS as the second dimension. The main band of protein which separated in the first dimension was demonstrated to be the PS I complex. This complex retained the intrinsic photochemical activity of P700 even after it was subjected to one-dimensional PAGE. These results suggest that certain protein complexes can be separated, with the maintenance of their original structures, by electrophoresis in the presence of the AES-DDAO mixture, and this method appears to have valuable potential for analysis of the components of membrane-bound protein complexes.     

水杨酸作用下东北红豆杉细胞的二维凝胶电泳分析

东北红豆杉悬浮培养体系中加入适量浓度的水杨酸,染色结果表明,水杨酸可以增加细胞膜通透性,并可诱导部分细胞发生核凝集或核碎裂。提取细胞染色体DNA进行琼脂糖凝胶电泳,发现染色体DNA发生了部分降解。应用蛋白质双向凝胶电泳技术,研究了水杨酸处理后东北红豆杉细胞发生应激反应过程中蛋白质的表达情况,分析了处理细胞与正常细胞的蛋白质组差异,发现在水杨酸处理48h后的样品中有7个蛋白质差异点,而且有6个蛋白点仅在对照中检测到。结果表明,外加水杨酸改变了东北红豆杉细胞的基因表达,抑制部分蛋白合成的同时也合成了部分新蛋白质,这些蛋白可能与水杨酸的作用有关。     

Electrophoretic and Immunological Properties of Folate-binding Protein Isolated from Bovine Milk

Changes of the folate-binding protein (FBP) concentration in bovine milk after parturition were investigated. The FBP was highly purified from mature milk by affinity chromatography. The purified FBP showed a single protein band in polyacrylamide gel electrophoresis and was immunologically homogenous in double immunodiffusion. However, in two-dimensional gel electrophoresis, the FBP was separated into several spots in isoelectric focusing in the first dimension, and each spot also showed two molecular weights in SDS-gel electrophoresis in the second dimension. But these FBP molecules were immunologically identical with each other. The neuraminidase treatment obviously diminished the number of isoelectric points of the FBP. Thus, the variety of FBP molecules was at least partially due to the variability of the sialic acid content in the carbohydrate moieties. Moreover, the milk FBP showed species-specificity among mammals immunologically as well as physicochemically.     

Changes in the haemolymph protein pattern during ovarian maturation in Xylocopa litipes

The haemolymph proteins of a hymenopteran insect Xylocopa litipes have been fractioned by the polyacrylamide gel disc electrophoresis. The haemolymph protein fractions have been examined histochemically. The changes taking place in the haemolymph protein pattern during vitellogenesis have been studied. Common protein fractions were observed in the haemolymph, fat body, ovary and testis. The role of sex specific protein during the vitellogenesis has been discussed.     

Identification of Cultivar Differences in Seed Polypeptide Composition of Peanuts (Arachis hypogaea L.) by Two-Dimensional Polyacrylamide Gel Electrophoresis

Seed polypeptides from several cultivars of peanut (Arachis hypogaea L.) have been compared by means of a two-dimensional polyacrylamide gel electrophoresis. Protein was extracted from the defatted peanut meal by homogenizing in 5 millimolar K₂CO₃-9.5 molar urea. After addition of Nonidet P-40 (2%, v/v) and dithiothreitol (0.5%, w/v) the solution was centrifuged at 25,000 g. This procedure led to solubilization of more than 95% of the total protein. The clear supernatant fraction was then subjected to two-dimensional polyacrylamide gel electrophoresis, employing isoelectric focusing in the first dimension and electrophoresis in presence of sodium dodecyl sulfate in the second. After examining several cultivars, it was possible to construct a composite map to include all of the polypeptide species found among all of the cultivars examined. At least 74 major and between 100 and 125 minor components were detectable by Coomassie blue staining. The majority of these had isoelectric points between pH 4.4 and 8.0, and molecular weights between 16,000 and 75,000. Several different cultivars have been compared using this method and it has been shown that considerable variation exists among the major polypeptides present. The method should prove valuable for analyzing different genotypes and selecting varieties with a particular storage protein make-up, as well as for following compositional changes that occur during seed development and germination.     

Stimulation and selective inhibition of protein synthesis by 17 beta-estradiol, in the uterus of immature rats

The in vitro translation of RNAs extracted from immature female rat uteri 24 hrs. after one or four daily injections of 17 beta-oestradiol was carried out. The proteins labelled with (35S) methionine were separated by polyacrylamide gel electrophoresis and the resulting autoradiograms were scanned. The densitometric profile of uterine proteins obtained after a single injection of oestrogen showed a considerable increase in the majority of them, compared with controls. Following four injections of 17 beta-oestradiol the synthesis of all but one of the uterine proteins was observed. The Mr of the protein induced by repeated administration of oestrogens was about 105 kdaltons. This protein could possibly be involved in the inhibition of protein synthesis observed in the rat uterus after reiterated injections of 17 beta-oestradiol to female rats.     

Author index for volume 202

A procedure is described for a simple two-step purification of human liver propionyl-CoA carboxylase. The method is based on acid and carbon tetrachloride extraction to remove other biotin carboxylases followed by an 800-fold purification through biotin-pretreated, monomeric avidin-Sepharose 4B-CL with elution of active enzyme using a biotin gradient. The enzyme had a sedimentation coefficient of 17.4 S and polyacrylamide gel electrophoresis after reduction and alkylation revealed two nonidentical polypeptide chains of 75,000 and 60,000 M_r. The heavier chain was identified as the biotin-containing subunit by electrophoresis after avidin binding.     

Abnormal protein patterns in multiple sclerosis

Sera from patients with multiple sclerosis (MS) and those obtained from normal subjects are indistinguishable by regular 5% or 7% polyacrylamide gel electrophoresis. However, 11 out of 15 MS sera examined by gradient polyacrylamide gel electrophoresis showed three distinct protein bands. None of the sera obtained from 10 normal subjects showed the characteristic protein patterns when they were examined by gradient gel electrophoresis. Similar results were obtained with de-albumin serum samples or with serum proteins precipitable at 50% ammonium sulfate saturation. These three proteins have now been purified to homogeneity by preparative gradient gel electrophoresis. Molecular weights of these proteins were estimated from gradient gel electrophoresis as 398,000, 363,000, and 302,000 daltons, respectively.This work was presented at the Tenth Annual Meeting of American Society for Neurochemistry on March 12, 1979, in Charleston, South Carolina.     

Analysis by two-dimensional polyacrylamide gel electrophoresis of the in vivo phosphorylation of ribosomal proteins derived from free and membrane-bound polysomes

Analysis of in vivo phosphorylation of mouse liver ribosomal proteins was performed by two-dimensional polyacrylamide gel electrophoresis following 32P-injection. Our method is special and differs from other eukaryotic systems reported in that all proteins separated on the first dimension gel are completely solubilized, moving quantitatively to the second dimension gel. Only ribosomes from polysomes were used, ensuring analysis of ribosomes actively engaged in protein synthesis. We resolved sixty-five distinct proteins from ribosomes from membrane bound or free polysomes. In both cases radioautography revealed similar labeled patterns with one highly phosphorylated ribosomal protein and five marginally labeled spots.     

Two dimensional benzyldimethyl-n-hexadecylammonium chloride----sodium dodecyl sulfate preparative polyacrylamide gel electrophoresis: a high capacity high resolution technique for the purification of proteins from complex mixtures

A two dimensional preparative polyacrylamide gel electrophoresis system is described which has both high resolving power and high loading capacity (approximately 100 mg protein). The first dimension is electrophoresis toward the cathode at acid pH in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC), and the second dimension is electrophoresis toward the anode in the presence of sodium dodecyl sulfate. Proteins purified in this way have been sequenced and used successfully as antigens.     

Differences in Protein Patterns in Suspension Cultures of Taxus cuspidata Induced by Cerium

The changes in soluble proteins induced by Ce⁴⁺ were analyzed in suspension cultures of Taxus cuspidata using two-dimensional polyacrylamide gel electrophoresis. The ultrastructure of cells obviously changed at day 4 after addition of Ce⁴⁺. Large amount of nuclear DNA fragments of about 200 bp were observed. Thirteen protein spots were different between the cultures grown with and without Ce⁴⁺ at day 4 as well as at day 6 after addition of Ce⁴⁺. This revised version was published online in July 2006 with corrections to the Cover Date.     

A two-dimensional polyacrylamide gel electrophoresis (PAGE) system using sodium dodecyl sulfate-PAGE in the first dimension.

A two-dimensional gel electrophoretic system for the separation of cellular proteins is described. The system utilizes sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in the first dimension and polyacrylamide gel isoelectric focusing in the second dimension. The system offers a good starting point for many difficult protein separations requiring SDS.     

Heterogeneity of human erythrocyte band 3 analyzed by two-dimensional gel electrophoresis

Human erythrocyte membrane and purified band 3 were separated initially by isoelectric focusing and then examined in a second dimension by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Band 3 was segregated into three major bands whether the protein was contained within the membranes or was present in the isolated state. The isoelectric points of these major bands were 5.25, 5.35 and 5.70. Of chymotryptic fragments of band 3, the 60-kDa fragment was also separated into three major bands whose pI values were 4.75, 5.10 and 5.30. The multiplicity of band 3 appears to be due to different charges carried by the peptide(s) and is not ascribed to oxidation of band 3 during its preparation. Isoelectric points of the purified 60-kDa fragment were different from the pI values of the fragment coexisting with the complementary 35-kDa fragment, in which case the pI values were exactly the same as those of intact band 3. This suggests that these fragments interact tightly in situ even after being cleaved by chymotrypsin, and the tight interaction must still be present during electrophoresis in the first dimension.     

Separation of t-RNAs by two-dimensional polyacrylamide gel electrophoresis

A pH 5.8 polyacrylamide gel electrophoresis buffer is described. Electrophoresis in this MES-citrate system at pH 5.8 separates E. coli transfer RNAs into 15 bands using 15% acrylamide gels. Polyacrylamide gel electrophoresis in a second dimension at pH 8.3 further resolves E. coli t-RNAs into 20 spots.     

Characterization of sucrose-hydrolyzing enzymes of Zymomonas mobilis

Extracellular proteins of Zymomonas mobilis were analyzed by two-dimensional gel electrophoresis and protein maps drawn up. One of these proteins showed sucrose-hydrolyzing activity, as indicated by activity staining after polyacrylamide gel electrophoresis. It was purified from the extracellular extract of a glucose fermentation by polyacrylamide gel electrophoresis, using a two-step procedure. The molecular mass of the protein was 46 kDa and its isoelectric point 5.0. A rabbit antiserum was raised against this protein. As shown by immunoblotting, the same protein was present in extracellular extracts obtained from glucose, fructose and sucrose fermentations. A cross-reaction was also detected by immunoblotting, with a cellular protein of molecular mass 46 kDa present on the three carbon sources studied. However, activity staining was unsuccessful on gels after electrophoresis of these cellular extracts. The extracellular protein extract obtained from a fermentation run on glucose contained another sucrose-hydrolyzing protein of molecular mass 51 kDa and with an isoelectric point of 4.8. This protein was absent in fructose and sucrose fermentations but showed a positive reaction with the antiserum raised against the 46 kDa extracellular protein. Partially purified sucrose-hydrolyzing proteins also catalyzed transfructosylation reactions, suggesting that they could be of the levansucrase type.