Silver staining of high molecular weight proteins on large-pore polyacrylamide gels

Perret, Beat A.; Felix, R.; Furlan, Miha; Beck, Eugene A.

doi:10.1016/0003-2697(83)90134-3

Cited by 8 publications

(3 citation statements)

References 22 publications

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“…SDS/polyacrylamide-gel electrophoresis The purpose of performing SDS/polyacryl-amide-gel electrophoresis in 3.3% gels in this investigation was not to demonstrate homogeneity of the highly purified mucin preparations but to demonstrate freedom from lower-Mr contamination. In our hands, the SDS/polyacrylamide-gelelectrophoresis methods reported to be suitable for resolution of high-Mr macromolecules unfortunately either were not completely reproducible (Perret et al, 1983) or were found to be inapplicable (Peacock & Dingman, 1968). When the crude soluble mucus was subjected to SDS/polyacrylamide-gel electrophoresis and stained with Coomassie Blue or Kenacid R, at least six bands, with Mr values <56000 (one band), 62000, 105000, 136000, 280000 and > 280000 (one band) respectively, were detectable (results not shown).…”

Section: Ultracentrifugal Analysismentioning

confidence: 78%

Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep

Mukkur¹,

Watson²,

Saini³

et al. 1985

Biochemical Journal

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Crude soluble mucus from sheep small intestine was freed of nearly all the nucleic acid contaminants by precipitation with protamine sulphate and treatment with nucleases. After removal of non-covalently bound proteins by equilibrium density-gradient centrifugation in CsCl, a high-Mr glycoprotein was isolated by repeated h.p.l.c. from the partially purified mucin. The high degree of purity of the high-Mr mucin was borne out by (a) the observation of a single boundary on analytical ultracentrifugation in the presence of 5M-guanidinium chloride and (b) the observation of apparent monodispersity on sedimentation-equilibrium analysis. The Mr of the highly purified mucin, determined by sedimentation equilibrium, was 5.0 (+/- 0.1) X 10(6) and was concentration-independent. Finally, only goblet cells and the mucus blanket lining the intestinal epithelial cells were immunofluorescent when guinea-pig anti-(highly purified mucin) serum was used in an indirect immunofluorescence assay. The above antiserum reacted with apparently equal strength with goblet cells and with free mucin in abomasum, caecum and colon. The chemical composition of the glycoprotein was 66% carbohydrate and 34% protein, 45% of the latter being composed of valine and threonine. The glycoprotein migrated anodally on immunoelectrophoresis and contained 7.1% (w/w) sulphate. Neutral hexoses accounted for nearly half of the total carbohydrate content, followed by galactosamine and glucosamine. Whereas fucose and sialic acid were present in only small amounts, uronic acid was not detectable in the highly purified mucus glycoprotein.

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Section: Ultracentrifugal Analysismentioning

confidence: 78%

Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep

Mukkur¹,

Watson²,

Saini³

et al. 1985

Biochemical Journal

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“…The sensitivity level of the latex assay (0.005 units antigen/ml) surpasses that of the common EIA by one order of magnitude. The antibody-coated latex particles have not changed within L6 months when stored at 4" C. Purified high-molecular weight FVIII/vWF and its fragments derived by disulfide reduction were electrophoresed on large-pore polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS) as recently described (6). The median number of dimeric subunits was estimated from the densitometric scans of Coomassie blue-stained gels.…”

Section: Lntroductionmentioning

confidence: 99%

Reactivity of Small Molecular Forms of Human Factor VIII/von Willebrand Factor with Botrocetin and Anti-Factor VIII-Coated Latex Particles

1985

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SummaryTwo recently developed tests for measurement of factor VIII/von Willebrand factor (FVIII/vWF), i.e. platetelet agglutination by botrocetin and a kinetic latex antigen assay, were compared with ristocetin cofactor and electroimmunoassay, in respect with FVIII/vWF size-distribution. FVIII/vWF was measured in six cases of atypical von Willebrand’s disease (type II), in gel-filtered fractions of normal cryoprecipitate and in the course of depolymerization of purified normal FVIII/vWF by disulfide reduction. Small molecular forms of FVIII/vWF from normal and variant type II plasma, and those derived by disulfide reduction of high-molecular weight FVIII/vWF, showed remarkably decreased reactivity in ristocetin-, botrocetin- and latex-assay. We conclude that for botrocetin-induced platelet agglutination, as well as for agglutination of antibody-coated latex particles, multiple interactions with repeating subunits of FVIII/vWF are required. As a practical consequence, the combined measurement of FVIII/vWF by the latex test and electroimmunoassay provides a simple tool for discriminating between the classical von Willebrand’s disease and its variants.

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“…Electrophoresis in the presence of sodium dodecyl sulfate (SDS) was performed in cylindrical 2.5% poly acrylamide gels, cross-linked with 2.75% methylene bisacrylamide (w/v) [11], Prior to electrophoresis, the samples were dialyzed for 60 min at room tempera ture against 0.01 A/NaCl-0.01 M Tris-HCl (pH 7.4). Aliquots of samples (up to 250 pi) were applied on the gels following addition of 1/10 vol of 10% SDS in 8 M urea and 1/10 vol of 20% glycerol (v/v) in 0.05% bromphcr.ol blue (w/v).…”

Section: Sds-polyacrylamide Gel Electrophoresismentioning

confidence: 99%

Isolation of Small Molecular Forms of Factor VIII/von Willebrand Factor from Plasma

Perret¹,

Furlan²,

Beck³

1984

Pathophysiol Haemos Thromb

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Cryoprecipitated factor VIII/von Willebrand factor (FVIII/vWF), freed of fibrinogen by clotting with calcium and Defibrase®, was chromatographed on Sepharose CL-2B. Fractions containing lower-molecular-weight forms of FVIII/vWF comprised coprecipitated plasma proteins of similar molecular weights. The major contaminants, fibronectin and IgM, were removed by affinity chromatography on gelatin- and anti-IgM-agarose, respectively. Finally, pure low-molecular-weight FVIII/vWF protein was harvested in the void volume fraction of a Sepharose CL-6B column. The smallest multimers had the size of the tetramer of the basic subunit chain of FVIII/vWF.

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Silver staining of high molecular weight proteins on large-pore polyacrylamide gels

Cited by 8 publications

References 22 publications

Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep

Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep

Reactivity of Small Molecular Forms of Human Factor VIII/von Willebrand Factor with Botrocetin and Anti-Factor VIII-Coated Latex Particles

Isolation of Small Molecular Forms of Factor VIII/von Willebrand Factor from Plasma

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