scholarly journals Identification and Purification of SpecificPenicillium marneffei Antigens and Their Recognition by Human Immune Sera

1998 ◽  
Vol 36 (4) ◽  
pp. 949-954 ◽  
Author(s):  
L. Jeavons ◽  
A. J. Hamilton ◽  
N. Vanittanakom ◽  
R. Ungpakorn ◽  
E. G. V. Evans ◽  
...  
Keyword(s):  
Western Blotting ◽  
Gel Electrophoresis ◽  
Pathogenic Fungus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Sds Page ◽  
Molecular Masses

Disseminated infection with the dimorphic pathogenic fungusPenicillium marneffei is increasingly seen among patients with AIDS in southeast Asian countries. Previous studies have demonstrated the presence of humoral immune responses to this fungus in patient sera; we have confirmed this work using sera from P. marneffei-infected patients (n = 21) to develop Western blots of P. marneffei cytoplasmic yeast antigen (CYA). P. marneffei CYA was then partially purified by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Immunoenzyme development of the Western blots with pooled sera from patients with P. marneffei infection and with pooled sera from patients with aspergillosis (n = 20), candidiasis (n = 10), cryptococcosis (n = 9), and histoplasmosis (n = 11) revealed three antigens with relative molecular masses of 61, 54, and 50 kDa. These antigens were specifically recognized by the pooled sera from the P. marneffei-infected patients. The 61- and 54-kDa antigens were subsequently purified to homogeneity by preparative gel electrophoresis, and the 50-kDa antigen was partially purified by the same technique. N-terminal amino acid sequencing revealed that the 61-kDa antigen had a strong homology (87% identity) with the antioxidant enzyme catalase. The three antigens were then subjected to SDS-PAGE and Western blotting and to immunoenzyme development with individual patient sera; sera from 86% of P. marneffei-infected patients recognized the 61-kDa antigen, sera from 71% recognized the 54-kDa antigen, and sera from 48% recognized the 50-kDa antigen. These specifically recognized antigens are the first to be purified from P. marneffei and can be used either singly or in combination to detect antibody responses in a large percentage of individuals infected with P. marneffei.

scholarly journals Lupus autoantibodies target ribosomal P proteins.

1985 ◽  
Vol 162 (2) ◽  
pp. 459-471 ◽  
Author(s):  
K B Elkon ◽  
A P Parnassa ◽  
C L Foster
Keyword(s):  
Western Blotting ◽  
Lupus Erythematosus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ribosomal Subunit ◽  
Polyacrylamide Gel ◽  
Antibody Activity ◽  
Composite Gel ◽  
Molecular Masses

All nine SLE (systemic lupus erythematosus) sera with antiribosomal antibody activity targeted the same three ribosomal protein antigens, of molecular masses 38 and 17/19 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. One serum reacted with an additional protein of approximately kD. Ribosomal subunit fractionation by composite gel electrophoresis and sucrose density ultracentrifugation showed that these proteins were part of the large subunit. Isoelectric focusing in agarose, and two-dimensional polyacrylamide gel electrophoresis revealed that the antigens had pI between 4.5 and 6.5, but that the 17/19 kD antigens were more acidic than the 38 kD antigen. Similarities in the molecular masses, charges, as well as the presence of highly conserved crossreactive epitopes, failure to bind to carboxymethylcellulose at pH 4.2, and extractability of the 17/19 kD proteins by 400 mM NH4Cl-ethanol at 0 degrees C indicated that these antigens were analogous to the proteins P0 (38 kD) and P1/P2 (17/19 kD) described previously (25, 36). Co-identity was confirmed using reference antibodies and antigen. Although antibodies to these proteins were only found in 5-10% of more than 50 sera screened by radioimmunoassay or Western blotting, the selective production of antibodies to epitopes on three (out of a total of more than 80) ribosomal proteins may provide further clues to autoantibody induction of SLE.

scholarly journals Humoral Immune Responses to S-Layer-Like Proteins of Bacteroides forsythus

2003 ◽  
Vol 10 (3) ◽  
pp. 383-387 ◽  
Author(s):  
Masahiro Yoneda ◽  
Takao Hirofuji ◽  
Noriko Motooka ◽  
Koji Nozoe ◽  
Kayoko Shigenaga ◽  
...  
Keyword(s):  
Immune Responses ◽  
Gel Electrophoresis ◽  
Early Onset ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Healthy Control ◽  
Specific Antigens

ABSTRACT Bacteroides forsythus is one of the important periodontopathic bacteria, and this microorganism is known to have an S-layer outside the outer membrane. The S-layer-like antigens were recently isolated from B. forsythus, and they were found to be 270- and 230-kDa proteins in the envelope fraction. In this study, these proteins were confirmed to be specific to B. forsythus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and they were clearly recognized by sera from patients with adult and early-onset periodontitis in Western immmunoblot analysis. We compared the immunoglobulin G (IgG) responses against the purified S-layer-like antigen by enzyme-linked immunosorbent assay. IgG responses against this antigen were low in healthy control subjects, but they were significantly higher in subjects with adult and early-onset periodontitis. Together with the fact that the IgG responses against the crude extract of B. forsythus did not rise significantly in patients with periodontitis, S-layer-like proteins are considered to be specific antigens of B. forsythus and may play an important role in the progression of periodontitis.

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.

The αEC domain of human fibrinogen-420 is a stable and early plasmin cleavage product

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2297-2303 ◽  
Author(s):  
Dianne Applegate ◽  
Lara Stoike Steben ◽  
Kathe M. Hertzberg ◽  
Gerd Grieninger
Keyword(s):  
Gel Electrophoresis ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cleavage Product ◽  
The Other ◽  
Cross Linking ◽  
Human Fibrinogen ◽  
Western Blots ◽  
Sds Page

Abstract Human fibrinogen-420, (Eβγ)2, was isolated from plasma and evaluated for its ability to form clots and for its susceptibility to proteolysis. Clotting parameters, including cross-linking of subunit chains, of this subclass and of the more abundant fibrinogen-340 (βγ)2, were found to be similar, suggesting little impact of the unique EC domains of fibrinogen-420 on coagulation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmic digestion patterns revealed production from fibrinogen-420 of the conventional fibrinogen degradation products, X, Y, D, and E, to be comparable to that from fibrinogen-340 in all respects except the presence of at least 2 additional cleavage products that were shown by Western blot analysis to contain the EC domain. One was a stable fragment (ECX) comigrating with a 34-kd yeast recombinant EC domain, and the other was an apparent precursor. Their release occurred early, before that of fragments D and E. Two bands of the same mobility and antibody reactivity were found in Western blots of plasma collected from patients with myocardial infarction shortly after the initiation of thrombolytic therapy.

scholarly journals Structural Properties of Lipopolysaccharides fromRickettsia typhi and Rickettsia prowazekii and Their Chemical Similarity to the Lipopolysaccharide from Proteus vulgaris OX19 Used in the Weil-Felix Test

1998 ◽  
Vol 66 (3) ◽  
pp. 923-926 ◽  
Author(s):  
Ken-ichi Amano ◽  
Jim C. Williams ◽  
Gregory A. Dasch
Keyword(s):  
Fatty Acids ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteus Vulgaris ◽  
Chemical Similarity ◽  
Rickettsia Prowazekii ◽  
Sds Page ◽  
Molecular Masses ◽  
Specific Antigens

ABSTRACT The lipopolysaccharides (LPSs) isolated from typhus group (TG) rickettsiae Rickettsia typhi and Rickettsia prowazekii were characterized by chemical analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. LPSs from two species of TG rickettsiae contained glucose, 3-deoxy-d-manno-octulosonic acid, glucosamine, quinovosamine, phosphate, and fatty acids (β-hydroxylmyristic acid and heneicosanoic acid) but not heptose. The O-polysaccharides of these LPSs were composed of glucose, glucosamine, quinovosamine, and phosphorylated hexosamine. Resolution of these LPSs by their apparent molecular masses by SDS-PAGE showed that they have a common ladder-like pattern. Based on the results of chemical composition and SDS-PAGE pattern, we suggest that these LPSs act as group-specific antigens. Furthermore, glucosamine, quinovosamine, and phosphorylated hexosamine were also found in the O-polysaccharide of the LPS from Proteus vulgaris OX19 used in the Weil-Felix test, suggesting that they may represent the antigens common to LPSs from TG rickettsiae andP. vulgaris OX19.

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots

2007 ◽  
Vol 53 (4) ◽  
pp. 526-532 ◽  
Author(s):  
Priyanka D. Abeyrathne ◽  
Joseph S. Lam
Keyword(s):  
Membrane Proteins ◽  
Western Blotting ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bacterial Membrane ◽  
Nitrocellulose Membrane ◽  
Western Blots ◽  
Sds Page ◽  
Highly Hydrophobic ◽  
Effective Transfer

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS–PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2–12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

Analysis for Cerebrospinal Fluid Proteins by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

1992 ◽  
Vol 38 (10) ◽  
pp. 2008-2012 ◽  
Author(s):  
F Mashige ◽  
T Shimizu ◽  
S Iijima ◽  
A Ohkubo
Keyword(s):  
Cerebrospinal Fluid ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Apolipoprotein A ◽  
Csf Proteins ◽  
Sds Page ◽  
Molecular Masses

Abstract Cerebrospinal fluid (CSF) proteins with molecular masses of < 150,000 Da were identified by immunoblotting after two kinds of nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). With PAGE 1 (17-27% gradient gel), CSF proteins were clearly separated into seven to nine bands with molecular masses of 3000-67,000 Da; seven bands were identified as beta 2-microglobulin, lysozyme, prealbumin, free kappa and lambda chain, apolipoprotein A-I, glycoproteins, and albumin by immunoblotting. With PAGE 2 (10-20% gradient gel), proteins were clearly separated into 11-16 bands with molecular masses of 15,000-150,000 Da; 11 were identified as prealbumin, free kappa and lambda chain, apolipoprotein A-I, glycoproteins, albumin, alpha 1-antitrypsin, transferrin (separated into two bands), immunoglobulin fragments, haptoglobin, and IgG. We analyzed CSF samples collected from 81 patients with cerebrospinal signs by these SDS-PAGE methods and observed prominent bands in some cases.

The αEC domain of human fibrinogen-420 is a stable and early plasmin cleavage product

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2297-2303 ◽  
Author(s):  
Dianne Applegate ◽  
Lara Stoike Steben ◽  
Kathe M. Hertzberg ◽  
Gerd Grieninger
Keyword(s):  
Gel Electrophoresis ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cleavage Product ◽  
The Other ◽  
Cross Linking ◽  
Human Fibrinogen ◽  
Western Blots ◽  
Sds Page

Human fibrinogen-420, (Eβγ)2, was isolated from plasma and evaluated for its ability to form clots and for its susceptibility to proteolysis. Clotting parameters, including cross-linking of subunit chains, of this subclass and of the more abundant fibrinogen-340 (βγ)2, were found to be similar, suggesting little impact of the unique EC domains of fibrinogen-420 on coagulation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmic digestion patterns revealed production from fibrinogen-420 of the conventional fibrinogen degradation products, X, Y, D, and E, to be comparable to that from fibrinogen-340 in all respects except the presence of at least 2 additional cleavage products that were shown by Western blot analysis to contain the EC domain. One was a stable fragment (ECX) comigrating with a 34-kd yeast recombinant EC domain, and the other was an apparent precursor. Their release occurred early, before that of fragments D and E. Two bands of the same mobility and antibody reactivity were found in Western blots of plasma collected from patients with myocardial infarction shortly after the initiation of thrombolytic therapy.

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