Detection of selenoproteins in human cell extracts by laser ablation-ICP MS after separation by polyacrylamide gel electrophoresis and blotting

2012 ◽  
Vol 27 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Juliusz Bianga ◽  
Guillaume Ballihaut ◽  
Christophe Pécheyran ◽  
Zahia Touat ◽  
Hugues Preud'homme ◽  
...  
Keyword(s):  
Laser Ablation ◽  
Human Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Cell Extracts ◽  
Icp Ms

scholarly journals Operation of the CO Dehydrogenase/Acetyl Coenzyme A Pathway in both Acetate Oxidation and Acetate Formation by the Syntrophically Acetate-Oxidizing Bacterium Thermacetogenium phaeum

2005 ◽  
Vol 187 (10) ◽  
pp. 3471-3476 ◽  
Author(s):  
Satoshi Hattori ◽  
Alexander S. Galushko ◽  
Yoichi Kamagata ◽  
Bernhard Schink
Keyword(s):  
Gel Electrophoresis ◽  
Coenzyme A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Acetate Oxidation ◽  
Co Dehydrogenase ◽  
Protein Patterns ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
Cell Extracts

ABSTRACT Thermacetogenium phaeum is a homoacetogenic bacterium that can grow on various substrates, such as pyruvate, methanol, or H2/CO2. It can also grow on acetate if cocultured with the hydrogen-consuming methanogenic partner Methanothermobacter thermautotrophicus. Enzyme activities of the CO dehydrogenase/acetyl coenzyme A (CoA) pathway (CO dehydrogenase, formate dehydrogenase, formyl tetrahydrofolate synthase, methylene tetrahydrofolate dehydrogenase) were detected in cell extracts of pure cultures and of syntrophic cocultures. Mixed cell suspensions of T. phaeum and M. thermautotrophicus oxidized acetate rapidly and produced acetate after addition of H2/CO2 after a short time lag. CO dehydrogenase activity staining after native polyacrylamide gel electrophoresis exhibited three oxygen-labile bands which were identical in pure culture and coculture. Protein profiles of T. phaeum cells after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the strain exhibited basically the same protein patterns in both pure and syntrophic culture. These results indicate that T. phaeum operates the CO dehydrogenase/acetyl-CoA pathway reversibly both in acetate oxidation and in reductive acetogenesis by using the same biochemical apparatus, although it has to couple this pathway to ATP synthesis in different ways.

Immunochemical reactivity of simian sarcoma virus polypeptides isolated by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis

1981 ◽  
Vol 27 (2) ◽  
pp. 238-242
Author(s):  
James C. Johnson ◽  
Tomoko Higuchi ◽  
Edwin Geissler
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Cell Extracts ◽  
Large Yield ◽  
Dodecyl Sulfate ◽  
Sarcoma Virus ◽  
Simian Sarcoma Virus

The polypeptides associated with a zonal centrifugation purified simian sarcoma virus propagated in lymphoblastoid NC-37 cells were isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) using a procedure designed to minimize the loss of immunochemical reactivity. The proteins p10, p15, p28, p36, p44, p75, and p86 were obtained in large yield and high degree of homogeneity. The electrophoretically purified p28 was analyzed by competition radioimmunoassay using antiserum to a pore exclusion and ion exchange purified simian sarcoma virus p28. Complete competition was observed with extracts of simian sarcoma virus infected cells. No competition was observed with uninfected or unrelated, infected cell extracts. The antigen–antibody affinity as measured by the slope of the competition curve using antiserum to p28 and 125I-labeled and electrophoretically purified p28 was the same as that for the p28 released from sonication-disrupted simian sarcoma virus. The data indicates that preparative purifications by polyacrylamide gel electrophoresis in the presence of SDS may be generally applicable for the isolation of proteins with essentially the same immuno-specificities and affinity for a specific antiserum as proteins isolated by procedures that avoid the use of SDS and electrophoresis.

4. The effect of interferon on the expression of human cell-surface antigens

1982 ◽  
Vol 299 (1094) ◽  
pp. 133-135 ◽  
Keyword(s):  
Human Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Antigens ◽  
Polyacrylamide Gel ◽  
Lymphoid Cells ◽  
Human Interferon ◽  
Interferon Α ◽  
Pulse Labelling ◽  
Hla Molecules

We have studied the effect of highly purified human interferon-α (the NK2-IFN preparation of Secher & Burke (1980)) on the expression of surface antigens by the human cell line Molt 4. Cytofluorimetric analysis with monoclonal antibodies revealed an increase in the membrane expression of HLA-A, B and C but not of other surface antigens. This effect has previously been observed with other human lymphoid cells (Fellous et al. 1979; Heron et al . 1968). The increase in the binding of fluorescent antibody is due to an increase in the number of HLA molecules in the membrane. This was demonstrated in an experiment in which surface proteins were iodinated after treatment with interferon. The difference in the content of HLA molecules could then be visualized on polyacrylamide gel electrophoresis of the immuno-purified surface antigens. In addition, a band corresponding to 16000 M r protein, absent from the surface of several human cell lines, has been found to be induced after IFN treatment. The effect of IFN-α on the synthesis of HLA and β2m was studied by pulse-labelling treated and untreated cells with [ 35 S]methionine for 3 h. The cells were then lysed, and the lysates used for immunoprecipitation with monoclonal antibody for HLA and with rabbit anti-human β2m serum. The immunoprecipitates were then analysed by SDS-polyacrylamide gel electrophoresis. A several-fold increase in the amount of newly synthesized HLA and β2m molecules was clearly observed. These results demonstrate that it is the rate of synthesis of HLA and β2m molecules that has been affected by the interferon treatment, thus leading to the enhanced expression of the antigens on the membrane, and to an increase in the secretion of free β2m.

Ultrastructural localization of specific nucleoprotein fractions

1978 ◽  
Vol 36 (2) ◽  
pp. 204-205
Author(s):  
G. L. Brown
Keyword(s):  
Gel Electrophoresis ◽  
Mouse Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ultrastructural Localization ◽  
Polyacrylamide Gel ◽  
Acid Extraction ◽  
Acid Solutions ◽  
Low Salt ◽  
Liver Nuclei ◽  
Phosphate Groups

Bismuth (Bi) stains nucleoproteins (NPs) by interacting with available amino and primary phosphate groups. These two staining mechanisms are distinguishable by glutaraldehyde crosslinking (Fig. 1,2).Isolated mouse liver nuclei, extracted with salt and acid solutions, fixed in either formaldehyde (form.) or gl utaraldehyde (glut.) and stained with Bi, were viewed to determine the effect of the extractions on Bi stainina. Solubilized NPs were analyzed by SDS-polyacrylamide gel electrophoresis.Extraction with 0.14 M salt does not change the Bi staining characteristics (Fig. 3). 0.34 M salt reduces nucleolar (Nu) staining but has no effect on interchromatinic (IC) staining (Fig. 4). Proteins responsible for Nu and glut.- insensitive IC staining are removed when nuclei are extracted with 0.6 M salt (Fig. 5, 6). Low salt and acid extraction prevents Bi-Nu staining but has no effect on IC staining (Fig. 7). When nuclei are extracted with 0.6 M salt followed by low salt and acid, all Bi-staining components are removed (Fig. 8).

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

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