Radiolabelling of the excretory-secretory and somatic antigens of Anisakis simplex larvae

1992 ◽  
Vol 66 (4) ◽  
pp. 305-309 ◽  
Author(s):  
Kazuo Sugane ◽  
Shu-Han Sun ◽  
Tadashi Matsuura
Keyword(s):  
Molecular Weight ◽  
Staphylococcus Aureus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anisakis Simplex ◽  
Molecular Weights ◽  
Human Sera ◽  
Somatic Antigens

ABSTRACTAnisakis simplex larvae were cultured in vitro in medium containing 35-methionine for ten days. The medium and the larval tissues were analysed for biosynthetically labelled polypeptide by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Immunoprecipitates with positive and negative human antisera were similarly analysed, using Staphylococcus aureus to absorb immunocomplexes. ES products of Anisakis larvae contained many polypeptides with molecular weights of less than 200 K. 180 KDa and 40 KDa polypeptides in ES products reacted with IgG in Anisakis-infected human sera. Somatic extracts also contained many polypeptides with molecular weights of less than 200 K. One of these polypeptides with a molecular weight of 130 K reacted with IgG in Anisakis-infected human sera. These polypeptides did not react with other nematode-infected human sera.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

scholarly journals Purification and subunit structure of legumin of Vicia faba L. (broad bean)

1974 ◽  
Vol 141 (2) ◽  
pp. 413-418 ◽  
Author(s):  
David J. Wright ◽  
Donald Boulter
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Broad Bean ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Subunit Structure ◽  
Isoelectric Precipitation ◽  
Molecular Weights

Zonal isoelectric precipitation was shown to be an effective method for the preparation of legumin which was homogeneous as judged by ultracentrifugation and polyacrylamide-gel electrophoresis. The subunit structure of legumin was investigated by preparative sodium dodecyl sulphate–polyacrylamide-gel electrophoresis and ion-exchange chromatography in urea. Five distinct subunits, of which two were acidic (α) and had a molecular weight of 37000, and three were basic (β) with molecular weights of 20100, 20900 and 23800, were identified. The α and β subunits were present in equimolar amounts in the legumin molecule and, in view of this and molecular-weight considerations, an α6β6 subunit model was proposed for legumin.

scholarly journals New Exfoliative Toxin Produced by a Plasmid-Carrying Strain of Staphylococcus hyicus

1999 ◽  
Vol 67 (8) ◽  
pp. 4014-4018 ◽  
Author(s):  
Hisaaki Sato ◽  
Takao Watanabe ◽  
Yasuko Murata ◽  
Ayumi Ohtake ◽  
Mayumi Nakamura ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Staphylococcus Aureus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
The Other ◽  
Exfoliative Toxin ◽  
Sds Page ◽  
Serotype B

ABSTRACT A new serotype of Staphylococcus hyicus exfoliative toxin (SHET), serotype B, was isolated from the culture filtrate of a plasmid-carrying strain of S. hyicus. The new SHET was purified by precipitation with 70% saturated ammonium sulfate, gel filtration on a Sephadex G-75 column, column chromatography on DEAE–Cellulofine A-500, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The new SHET caused exfoliation of the epidermis as determined by the so-called Nikolsky sign when inoculated into 1-day-old chickens. The new SHET was serologically different fromStaphylococcus aureus exfoliative toxins (ETs) (ETA, ETB, and ETC) and from the SHET from the plasmidless strain but showed the same molecular weight as the other serotypes of toxins on SDS-PAGE. It was thermolabile and lost its toxicity after being heated at 60°C for 30 min. We propose that the new SHET be designated SHETB and that the SHET produced by the plasmidless strain be designated SHETA.

Isoelectric points and molecular weights of salt-extractable ribosomal proteins

1976 ◽  
Vol 54 (12) ◽  
pp. 1029-1033 ◽  
Author(s):  
M Saleem ◽  
Burr Atkinson
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Isoelectric Points ◽  
Acidic Proteins ◽  
Gel Isoelectric Focusing ◽  
Liver Ribosomes

Rat liver ribosomes prepared in low salt buffer contain basic and acidic proteins not found on ribosomes washed in high salt buffer. Proteins extracted from liver ribosomes by 500 mM KCl were characterized by acid urea–polyacrylamide gel electrophoresis, sodium dodecyl sulfate – polyacrylamide gel electrophoresis and gel isoelectric focusing. The salt-solubilized proteins contain 12 polypeptides with a molecular weight over 67 000, several polypeptides with molecular weights less than 67 000, and three polypeptides whose molecular weight exceeded 130 000. Ten to 12 of the proteins were basic, and about 24 acidic proteins were partially or wholly extracted from the ribosomes. Four of the acidic proteins have isoelectric points less than 4.5.

scholarly journals Incorrect Molecular Weights due to inaccurate Prestained Protein Molecular Weight Markers that are used for Gel Electrophoresis and Western Blotting

2020 ◽  
Author(s):  
Rômulo Leão Silva Neris ◽  
Ajuni Kaur ◽  
Aldrin V. Gomes
Keyword(s):  
Molecular Weight ◽  
Molecular Mass ◽  
Western Blotting ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Molecular Weights ◽  
Molecular Masses ◽  
Protein Molecular Weight

ABSTRACTThe most widely used Western blotting protein standards are prestained proteins of known molecular mass (kDa). They are also utilized for sodium dodecyl sulphate (SDS) Polyacrylamide Gel Electrophoresis (PAGE) to determine the molecular mass of proteins separated by electrophoresis. The objective of this study was to assess the reliability of different commercially available protein standards in predicting accurate protein molecular weights. We performed this experiment by running Criterion TGX gels with five prestained protein standards (Thermo Fisher SeeBlue Plus 2, Bio-Rad Precision Plus Protein Dual-color, Thermo Fisher Spectra Multi-color, Novex-Sharp Pre-stained, and Invitrogen iBright Pre-Stained). To evaluate their accuracy, we utilized highly purified Bovine Serum Albumin (BSA, 66.44 kDa) and Cytochrome C (Cyto C, 11.62 kDa). We also made use of the dimers of BSA (132.88 kDa) and Cyt C (23.24 kDa) that are present on SDS-PAGE gels. Our results suggest that three of the standards were less accurate at higher molecular masses with the iBright marker having the highest error in determining the expected 132.88 kDa molecular weight. The SeeBlue Plus 2 was accurate at identifying the 132.88 kDa molecular weight protein band but was less reliable for the three other lower molecular weight proteins. These findings have significant implications for the determination of protein masses because researchers rely on these standards to evaluate the molecular masses of their protein(s). We suggest that at least two different protein standards should be initially used in electrophoresis gels and for Western blotting in order to get accurate protein molecular weight results.

Analysis of excretory–secretory and somatic antigens ofGastrothylax crumenifer

2000 ◽  
Vol 74 (3) ◽  
pp. 271-276 ◽  
Author(s):  
M.K. Saifullah ◽  
Gul Ahmad ◽  
W.A. Nizami
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Potential Importance ◽  
Molecular Weights ◽  
Tissue Extracts ◽  
Gastrothylax Crumenifer ◽  
Metabolic Products ◽  
Somatic Antigens ◽  
Somatic Extract

AbstractThe excretory/secretory (ES) metabolic products released byGastrothylax crumenifer(Trematoda: Digenea) duringin vitroincubations and the somatic extract of the adult parasite were analysed using polyacrylamide gel electrophoresis (PAGE). Immunogenicity of ES and somatic extracts were evaluated by immunoblotting and ELISA using sera raised against ES and somatic antigens in rabbits. The electropherograms of ES and somatic extracts have been resolved into 38 and 41 polypeptides, respectively. The apparent molecular weights of these polypeptides range from < 29 to > 205 kDa. A total of 14 polypeptides were found to be common to both of the samples. The analysis of immunoblot results revealed 22 and 27 antigenic polypeptides in ES and somatic extracts respectively. Only 11 and 13 antigenic polypeptides were found specific to ES and tissue extract respectively. The molecular weights of these specific polypeptides were calculated to be < 14.4, 16, 20, 25, 33, 42, 119, 125 and > 205 kDa for metabolic products and < 14.4, 25, 30, 35, 78, 84 and > 205 kDa for the tissue extracts, respectively. Analysis of ELISA results revealed that a dilution of up to 1:3200 of the test sera could react with the ES product. Further, when the ES antigens were allowed to react with antisomatic extracts in hyperimmune sera the titre of IgG increased up to a dilution of 1:12800. The potential importance of these antigens in the immunodiagnosis of amphistomiasis is discussed.

scholarly journals Induction by phenobarbital of the mRNA for a specific variant of rat liver microsomal cytochrome P-450

1981 ◽  
Vol 196 (3) ◽  
pp. 839-851 ◽  
Author(s):  
I R Phillips ◽  
E A Shephard ◽  
F Mitani ◽  
B R Rabin
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Microsomal Cytochrome ◽  
Liver Cytochrome ◽  
Cytochrome P 450 ◽  
Induction By Phenobarbital

The treatment of rats for 4 days with phenobarbital causes an apparent 3-fold increase in the amount of total liver cytochrome P-450. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, metyrapone binding and immunoprecipitation, this increase was found to be due to a much larger increase in a restricted number of specific cytochrome P-450 variants. A radioimmunoassay technique demonstrated that the major phenobarbital-inducible variant, of molecular weight 52 000, is induced 24-fold by phenobarbital. Immunoprecipitation analysis of products of translation in vitro with an antibody specific to the 52 000-mol.wt. cytochrome P-450 showed that phenobarbital induces the mRNA in polyribosomes for this variant 20-fold. Evidence is presented for the action of phenobarbital at the transcriptional and translational levels.

Effect of testicular hormones on synthesis of soluble proteins by dog prostate slices

1983 ◽  
Vol 61 (7) ◽  
pp. 756-763 ◽  
Author(s):  
Jean Y. Dubé ◽  
Pierre Chapdelaine ◽  
Roland R. Tremblay
Keyword(s):  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Blue Staining ◽  
Secretory Proteins ◽  
Molecular Weights ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

We have submitted adult mongrel dogs to various endocrine manipulations. Prostate slices from these animals were then incubated in vitro in the presence of [3H]leucine or [35S]methionine. We have analyzed the cytosolic proteins by polyacrylamide gel electrophoresis. In intact adult uncastrated dogs, the radioactive amino acids were incorporated into three major bands having respective molecular weights (MW) of 32 000,16 000, and 15 000 in one-dimensional gels in presence of sodium dodecyl sulfate and mercaptoethanol. Two-dimensional electrophoresis revealed heterogeneity of each of these bands, both in isoelectric focussing (IEF) or nonequilibrium pH gel electrophoresis (NEpHGE) conditions. The 32 000 MW proteins showed five to six major radioactive spots and the 15 000 – 16 000 MW proteins showed six to seven spots by IEF. However, the highest incorporation of radioactivity occurred in a 16 000 MW protein seen only in NEpHGE. The lower MW proteins corresponded to some of the major proteins of dog seminal plasma as observed by immunoprecipitation of prostate proteins with antibodies against whole seminal plasma. By contrast, the 32 000 MW proteins were minor proteins of prostate cytosol and seminal plasma by Coomassie blue staining. Castration for 2 weeks completely abolished the synthesis of all these proteins. When castrated animals were treated with 5α-androstane-3α-17β-diol (10 mg/day for 2 weeks), the pattern of protein synthesis returned to the one observed in intact uncastrated animals. These observations show that testicular androgens control the synthesis of dog prostate major secretory proteins.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

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