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PubMed1985

Membrane proteins in Rh+, Rh- and Rh: -29, 38 (- - -/- - -, rh) red cells compared by using SDS-polyacrylamide gel electrophoresis.

Since Rh: -29, 38 (- - -/- - -, rh) phenotype of the Rh blood groups (--- in text) revealed unusual red cells, such as stomatocytes and microspherocytes and the relatively shortened half life of 17 days, red cell membrane proteins from Rh + (D), Rh - (d) and --- were compared by using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). No differences were observed among the patterns of the reduced and non-reduced membrane proteins from Rh+, Rh- and --- red cells. Two-dimensional gel electrophoresis of --- red cell membrane proteins also revealed a pattern similar to Rh+ and Rh- red cell membrane proteins. It is suggested that the lack of all Rh antigens causes no visible alteration of red cell membrane proteins detected by the method of Fairbanks G., Steck T.L. and Wallach D.F.H. (1971) Biochemistry, N.Y. 10, 2606-2617.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Renaturation of Leishmania donovani 3'-nucleotidase following sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. Renaturation of a 3'-nucleotidase from the surface membrane of Leishmania donovani promastigotes was achieved following polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). 2. Enzyme activity was detected in situ in gels, following SDS removal, by incubating the gels in reaction mixtures containing 3'-AMP or 3'-UMP as substrate followed by staining for the inorganic phosphate (Pi) reaction product with malachite green-molybic acid solution. 3. Conditions for the removal of SDS by diffusion and for the renaturation of enzyme activity are described including evidence for the detergent requirement, which is best satisfied by 3[(3-cholamidopropyl)-dimethylammonio]2-hydroxy-1-propane sulfonate (CHAPSO). 4. Results indicate that the 3'-nucleotidase migrates under these conditions as a polypeptide with an Mr of 43,000.

Animals↗
PubMed1989

Lysozyme activity in animal extracts after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. Lysozyme activity was detected after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels containing 0.2% (W/V) autoclaved Micrococcus lysodeikticus cells as substrate. 2. Lysozyme activity appeared as clear lysis zones after incubation of opaque gels at 37 degrees C in buffered Triton X-100. 3. As low as 0.1 pg of purified hen egg white lysozyme could be detected after 16 hr incubation at pH 6.5. 4. Bands with lytic activity from kidney and pancreas acetone powders, bird's egg whites and vitelline membranes, animal sera and human saliva corresponded to c-type (Mr 14,500), g-type (Mr 20,500) or both lysozymes as far as molecular weight is concerned. 5. Some extracts, like porcine kidney, exhibited more than two bands. 6. Bands with lytic activity migrating at the level of g-type lysozymes were detected in some kidney and pancreas extracts.

Animals↗
PubMedDate not supplied

River buffalo (Bubalus bubalis L.) AA phenotype haemoglobins: characterization by immobiline polyacrylamide gel electrophoresis and high performance liquid chromatography and determination of the primary structure of the constitutive chains by mass spectrometry.

1. Polyacrylamide gel electrophoresis in ultra-narrow immobilized pH gradient shifted the "Hb fast" band of AA buffalo phenotype haemoglobin into two components which were named Hb1 and Hb3. 2. Urea/Triton electrophoresis and reversed-phase HPLC demonstrated that Hb1 and Hb3 differ in the presence of two structurally distinct alpha chains (alpha 1 and alpha 3), also suggesting that the alpha chains must differ for neutral amino acid substitution. 3. Extensive mass spectrometric analysis on several digests (FAB overlapping) meant to determine the complete sequence of the constituent chains. 4. Two amino acid replacements (Lys 18----His and Asn 116----His) were present in the beta chain with respect to the bovine (A phenotype) chain, whereas the alpha 1 and alpha 3 globins were found to contain four amino acid replacements compared to the bovine alpha, three of which were identical (Glu 23----Asp, Glu 71----Gly and Phe 117----Cys) and, notably, an insertion of Ala at position 123-124. 5. Furthermore, alpha 1 contains Phe at position 130 whereas alpha 3 contains Ser at position 132 (following the modified numbering as a consequence of the Ala insertion).

Acrylamides↗
PubMed1980

SDS-polyacrylamide gel electrophoresis of isolated cortices of Xenopus laevis eggs.

Outer cytoplasmic membranes of Xenopus laevis eggs were isolated by manual dissection. Cortices of unfertilized eggs and membranes of fertilized stages were subjected to SDS-polyacrylamide gel electrophoresis. The Coomassie-blue stained gels of membranes from unfertilized eggs and fertilized stages showed 21 and 17 major bands, respectively, covering a molecular weight range of 27000 - 275000 Daltons. To demonstrate their purity, the preparations were examined by light and electron microscopy and the gel patterns were compared with those of gel electropherograms of isolated envelopes, yolk and pigment granules. To detect membrane proteins that reside in the outer membrane surface, the eggs were exposed to 131Iodine in the presence of lactoperoxidase. Approximately four proteins of molecular weights of 115000, 78000, 55000 and 27000 Daltons were labelled. In addition, all except one of the proteins of the vitelline envelope were strongly labelled, while the yolk platelets could only be iodinated after isolation. Changes of cortical protein patterns were studied in eggs and cortices: a) after fertilization; b) after activation by calcium-ionophore A 23187; c) after addition of calcium and distilled water to isolated cortices; and d) after incubation in the presence of the proteolytic enzyme papain. After fertilization, bands with molecular weights of 246000, 181000, 166000 and 34000 Daltons were missing. Ionophore and calcium treatment of unfertilized eggs produced a protein pattern similar to that observed after normal fertilization. After papain treatment, the band patterns of the membranes were not significantly different from those of cortices of unfertilized eggs.

Animals↗
PubMed1991

Optimal conditions for the analysis of Pasteurella haemolytica lipopolysaccharide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

The optimal conditions for the analysis of the lipopolysaccharide (LPS) of two serotype A1 isolates and a serotype A2 isolate of Pasteurella haemolytica by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining were determined. The LPS of the A1 isolates possessed O side chains, consisting of high molecular mass bands with the appearance of a ladder-like pattern, as well as a low molecular mass core-oligosaccharide region; the LPS of the A2 isolate consisted only of the core-oligosaccharide region. Furthermore, the LPS of the two A1 isolates differed in the core-oligosaccharide region. Optimal resolution of low molecular mass LPS components was obtained in a 15% acrylamide resolving gel containing 4 M urea whereas optimal resolution of high molecular mass components was obtained when urea was omitted. Conventional silver staining resulted in excellent visualisation of LPS bands, whereas a modified staining method did not detect additional bands, as has been demonstrated with the LPS of Pseudomonas aeruginosa. Proteinase K digestion of outer membranes gave more clearly defined LPS profiles than did similar digestions of whole cells, and more closely resembled the profiles of purified LPS. With the exception of slight variation in the average molecular mass of a group of O side chains between logarithmic and stationary phases there were no differences in LPS profiles at various stages of the growth cycle; freezing and thawing of LPS samples had no effect on the profiles.

Animals↗
PubMed1978

The isolation of DNA from agarose gels by electrophoretic elution onto malachite green-polyacrylamide columns.

Electrophoretic elution of DNA coupled with direct adsorption onto malachite green-polyacrylamide columns was used to isolate double- and single-stranded DNA from agarose gels. Subsequently, DNA was eluted with a high salt buffer and filtered through Sephadex which permitted recovery of the DNA in a low salt buffer at concentrations suitable for heteroduplex analysis by electron microscopy. This method was tested by examining heteroduplexes formed from the isolated complementary single strands of T7 wild type DNA and a T7 deletion mutant. More than 80% of the reannealed molecules were intact heteroduplexes showing the deletion loop. Irradiation of single-stranded DNA with 254 nm light resulted in distorted, convoluted heteroduplexes while 366 nm light did not show this effect.

Coliphages↗
PubMed1983

Identification of envelope and nucleocapsid proteins of infectious bovine rhinotracheitis virus by SDS-polyacrylamide gel electrophoresis.

Infectious bovine rhinotracheitis (IBR) virus was purified by rate zonal and isopycnic centrifugation in potassium tartrate gradients. Viral nucleocapsids were isolated from purified virions by treatment with the nonionic detergent Triton X-100 followed by high speed centrifugation. This treatment was shown to produce a suspension of 74% completely de-enveloped nucleocapsids, 24% incompletely de-enveloped nucleocapsids, and 2% whole virions. The viral nucleocapsids contained DNA and banded at a density of 1.25 g/cm3. Analysis of the viral polypeptides by gradient SDS-polyacrylamide gel electrophoresis revealed that 33 virion proteins, ranging in molecular weight from 13,000 to 275,000 dalton, were present in the complete virus particle. Detergent treatment of the virus quantitatively removed two of the major proteins (vp8, 90,000 dalton, and vp13, 73,000 dalton) and partially removed eleven other proteins. Fifteen viral polypeptides appeared to remain firmly associated with the viral nucleocapsids.

Animals↗
PubMed1985

A study of antigenic variants of foot-and-mouth disease virus by polyacrylamide gel electrophoresis of their structural polypeptides.

Twenty-nine foot-and-mouth disease (FMD) type A virus strains, previously classified serologically as distinct subtypes were analysed by polyacrylamide gel electrophoresis (PAGE) to determine the extent of variation in the pattern of the structural polypeptides and to evaluate the technique as an aid to existing subtyping techniques. The majority of the subtypes examined had distinct polypeptide patterns, however, some variation also occurred between strains within a subtype. The position of VP2(1B) and VP3(1C) was often unchanged in different strains within a subtype and between geographically related subtypes over long periods of time. Changes in the position of VP1(1D) were also observed within a subtype. The technique was considered to be of value for the screening of isolates prior to conventional serological subtyping procedures and in the tracing of the possible origin of FMD outbreaks.

Animals↗
PubMed1993

Survey of rotavirus infection in a dairy herd: comparison between polyacrylamide gel electrophoresis and two commercial tests.

A survey of rotavirus infection in a dairy herd with a history of neonatal diarrhoea was carried out. Faecal samples taken from 15 cows before and after calving as well as faeces taken from their calves daily from birth to two weeks of life were tested for rotavirus by polyacrylamide gel electrophoresis (PAGE) and compared with an ELISA and a latex agglutination commercial test. Rotavirus excretion was not detected in faeces from cows around parturition by any of the three tests. However, all of their calves shed rotaviruses during the observation period. The onset of rotavirus excretion determined by PAGE ranged from day 2 to day 8 of life (day 4.8 +/- 1.8 on average) and lasted for 4 to 7 days (5.3 +/- 1.1 days on average). Chi-square test showed a significant association (P = 0.0001) between the presence of rotavirus and the altered consistency of calves faeces. All the three tests showed similar results (overall agreement 92.5%) but discrepancies were detected mainly at the beginning or at the end of the rotavirus excretion period. Results obtained with both commercial kits closely paralleled each other and parameters other than sensitivity, specificity, diagnostic accuracy or predictive values have to be considered as selection criteria.

Animals↗
PubMed1994

Protein profiles of Brucella suis and Brucella abortus in isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

The protein profiles of five field isolates of Brucella suis and four field isolates of B. abortus were examined. Isoelectric focusing of soluble proteins from cell sonicates produced 28 bands common to both species, with a further 10 bands being unique to a species. No intraspecies variation was detected. Sodium dodecyl sulfate extracted proteins were resolved by polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose and then stained. These techniques allowed the demonstration of 35 bands which were common to both species, with a further 15 bands which were unique to one of the two species. Again, no intraspecies variation was detected.

Animals↗
PubMed1980

Comparison, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of urinary proteins excreted by workers exposed to cadmium, mercury or lead.

Urinary proteins from workers exposed to cadmium (Cd, n = 20), lead (Pb, n = 19) or mercury (Hg, n = 25) were analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The areas of the peaks with the relative mobilities of beta 2-microglobulin and retinol binding protein (low molecular weight proteins), albumins, transferrin and IgG (high molecular weight proteins) were measured and compared with those obtained in matched control groups. The concentrations (mean and range) of the metals in blood (B, micrograms/100 ml) and in urine (U, micrograms/g creatinine) of the exposed groups were as follows: Cd group, CD-B = 1.51 (0.18-4.31) and CD-U = 16.6 (2.08-59.3); Pb group, Pb-B = 44.5 (44.8-61.3) and Pb-U = 88.7 (15.2-298); Hg group, Hg-B = 3.74 (1.84-8.14) and Hg-U = 116 (51.8-206). The average duration of exposure to the metal was 30.5 (Cd), 12.5 (Pb) and 7.2 (Hg) years respectively. Under these exposure conditions, it was found that Cd increases the urinary excretion of low as well as high molecular weight proteins, Hg induces an increased urinary excretion of only high molecular weight proteins and lead has no significant effect on the renal excretion of either type of protein.

Cadmium↗
PubMed1987

Computer analysis of proteins separated by polyacrylamide gradient pore gel electrophoresis.

A method for measuring lymph-to-plasma (L/P) protein concentration ratios obtained from protein fractions separated by polyacrylamide gradient gel electrophoresis is presented. A curve-fitting technique is used to decompose lymph and plasma electropherograms containing multiple components into individual components, eliminating protein-protein overlap regions. This allows the concentration of each component in the mixture to be measured accurately, yielding more precise estimates of L/P ratios. This technique consists of three phases. Individual electropherograms are constructed for proteins of various sizes by taking a weighted average of measured electropherograms obtained from the two protein standards closest in size to the protein of interest. Using these generated standard curves, the multicomponent lymph and plasma curves are decomposed into the least number of equally spaced components that yield a good fit. A linear least-squares method is used to do this. Each protein fraction is multiplied by the total measured protein concentration to provide a concentration for each component. Finally, L/P concentration ratios of protein fractions with visible peaks were computed by applying an averaging technique to the equally spaced protein fractions. Plots of sheep lung L/P ratio versus protein size obtained in this manner were compared to L/P ratios obtained using a method of analysis which does not correct for protein overlap. The corrected L/P ratios showed less scatter than the uncorrected curves. Lung lymph data analyzed with the correction method indicated an increased lung microvascular permeability for large proteins following endotoxin infusion, whereas the uncorrected curves were too noisy to support this concept.

Animals↗
PubMed1991

Staining and quantification of proteins separated by polyacrylamide gel electrophoresis.

The present review concentrates on techniques for the staining and quantification of proteins separated by polyacrylamide gel electrophoresis. Staining with organic dyes has been used for approximately thirty years; the silver staining technique was introduced in 1979. The problems of silver staining are presented separately because the mechanism of this staining is in principle different from staining with organic dyes. Less attention has been devoted to quantification of two-dimensional gels, because this autoradiography is preferred because of its high sensitivity and fewer problems with accurate quantification in contrast to silver staining.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Silver staining of collagen type I after sodium dodecylsulphate polyacrylamide gel electrophoresis: effect of Maillard reaction.

Differences in the acidic silver staining, after sodium dodecylsulphate polyacrylamide gel electrophoresis, between the alpha 1 and alpha 2 collagen chains, as well as between rat-tail tendon and calf-skin collagen type I, were observed. The staining conditions at which the staining differences are both most expressed and reproducible were characterized. Age differences between staining of the alpha 1 CB6 fragment from young rats (2 and 12 months) and old rats (29 months) indicated that different susceptibilities of collagen species to the silver staining can be the result of different extents of some age-dependent post-translational modification, such as glycation. In vitro incubation of acid-soluble rat-tail tendon collagen with various sugars led to an increase in silver staining compared with samples incubated in the absence of sugar. This effect was inhibited by sodium cyanoborohydride, diethylenetriamine pentaacetic acid and aminoguanidine, i.e. compounds inhibiting the Maillard reaction at various stages. It can be concluded that the enhanced silver susceptibility of glycated collagen is related to advanced-phase Maillard reaction products attached to collagen.

Animals↗
PubMed1992

Improved procedure for the drying and storage of polyacrylamide slab gels.

An improved procedure for the drying and storage of polyacrylamide (PAA) slab gels of various sizes and acrylamide contents is presented. A PAA slab gel is preferentially fixed in methanol-acetic acid-glycerol-water (1.0:1.0:0.1:2.0) and then dried under low pressure in an adapted photoprint dryer between Cellophane and polyethylene foils at 75-80 degrees C for 50-90 min. Strong, transparent and flexible sheets are obtained that can be stored for extended periods. The potential of the procedure is exemplified following electrophoresis of both lipopolysaccharides and protein isolated from Coxiella burnetii bacterium.

Bacterial Proteins↗
PubMed1992

Isolation and purification of amyloid protein A by sodium dodecyl sulphate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography (HPLC) were used consecutively for the isolation of amyloid protein A (protein AA) from amyloid fibrils. Highly purified protein AA was obtained and determined by electrophoretic and amino acid analyses. The heterogeneity of protein AA was shown by HPLC. The isoforms of protein AA had different hydrophobicities, although they were equal in size and similar in amino acid composition. Compared with the conventional amyloid separation procedure (gel permeation chromatography), this technique is rapid, requires only small amounts of amyloid fibrils and may provide new information on amyloid proteins.

Amino Acids↗
PubMed1994

Immobilization of heparin on polyacrylamide derivatives.

Heparin was coupled via its carboxyl group with a polyacrylamide derivative containing covalently bound amino groups using the carbodiimide reaction. Heparin immobilized in this way proved to be useful as an affinity carrier for the isolation of antithrombin III and heparin-binding proteins from boar seminal plasma.

Acrylic Resins↗