Western immunoblotting: Temperature-dependent reduction in background staining

Thean, E; Toh, Ban Hock

doi:10.1016/0003-2697(89)90048-1

Cited by 26 publications

(5 citation statements)

References 14 publications

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“…Aliquots (10 or 50 lg) of crude extracts were fractionated by electrophoresis through a 12% SDS-polyacrylamide gel in a Mini PROTEAN II cell (Bio-Rad, Hercules, Calif., USA). BLOTTO (Thean and Toh 1989) was used as blocking reagent and for incubations with antibodies. Proteins were electroblotted onto a PVDF membrane (Roti-PVDF, Carl Roth, Karlsruhe, Germany).…”

Section: Western Analysismentioning

confidence: 99%

The stringent response, σH-dependent gene expression and sporulation in Bacillus subtilis

2001

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Recently, we found that transcription of the yvyD gene of Bacillus sibtilis from a sigmaH-dependent promoter is induced in response to amino acid starvation. This induction occurred in the wild type, but not in the relA mutant, suggesting that ppGpp might be involved in the activa tion of sigmaH. In order to substantiate this finding we looked for other sigmaH-dependent genes which also required an active relA gene product for activation in amino acid-starved cells. Three additional transcription units, namely spo0A, spoVG and the ytxGHI operon (also known as the csb40 operon), were found to be under the dual control of RelA and sigmaH in amino acid-starved cells, but many other sigmaH-dependent genes were not activated under these conditions. Of particular interest is the observation that spo0A is one of the genes that need a functional relA gene product for sigmaH-dependent gene activation. Furthermore, in sporulation medium, both Spo0A accumulation and phosphorylation are delayed in the relA deletion strain, leading to a delay in sporulation. A comparison between a relA point mutant and a relA deletion mutant revealed that the relA deletion mutant exhibits a more pronounced delay in the appearance of spores. These results suggest that the stringent response is not required for sporulation per se, but rather enhances the efficiency of sporulation.

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Section: Western Analysismentioning

confidence: 99%

The stringent response, σH-dependent gene expression and sporulation in Bacillus subtilis

2001

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“…If increased background tends to be a problem, the use of 1% BSA-C (Aurion, Wageningen, The Netherlands) in 5% appropriate normal serum can be very helpful. If the blocking reaction is performed in the cold instead of room temperature, the concentrations of the solutions may have to be adjusted, usually reduced Thean and Tho 1989).…”

Section: Blocking Of Unspecific Antibody-binding Sitesmentioning

confidence: 99%

Immunocytochemistry and in situ hybridization in the electron microscope: combined application in the study of virus-infected cells

Bienz

Egger

1995

Histochem Cell Biol

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The present review evaluates methods for electron microscopic immunocytochemistry and in situ hybridization, using post-embedding techniques and colloidal gold as a label. Special emphasis is given to double labeling immunocytochemistry and double in situ hybridization and to their combined application on the same specimen. Brief guidelines are presented for fixation, embedding media, the use of polyclonal and monoclonal antibodies and nucleic acid probes. Conditions for labeling and binding of antibody and nucleic acid probes to the target and protocols for direct and indirect immunodetection are discussed. Combinations of direct and indirect immunodetections in multiple labeling experiments are summarized.

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“…Nonidet P-40, and incubated for 60 min with horse-radish peroxidase-conjugated rabbit antimouse immunoglobulin (Dakopatts, Denmark; diluted 1 : 100 in blocking solution). All incubation steps were performed at 4°C to prevent non-specific binding [35]. The strips were again washed, rinsed in NaC1/Pi and antibody binding was detected by incubation with 0.05% (massivol.)…”

Section: Immunoblottingmentioning

confidence: 99%

Monoclonal antibodies specific for the core protein of the β‐subunit of the gastric proton pump (H⁺/K⁺ ATPase)

Jones

Toh

Pettitt

et al. 1991

European Journal of Biochemistry

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The gastric H+/K+-transporting adenosine triphosphatase ( H + / K + ATPase) (proton pump) consists of a catalytic a-subunit and a recently proposed 60 -90-kDa glycoprotein p-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60 -90-kDa glycoprotein.The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin-fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60 ~ 90-kDa molecule from tubulovesicular membranes and a 35-kDa component from peptide N-glycosidase-F-treated membrane extracts ; ( 5 ) immunoblotted the 60 ~ 90-kDa parietal cell autoantigen associated with autoimmune gastritis and pernicious anemia, purified by chromatography on parietal cell autoantibody-or tomato-lectin -Sepharose 4B affinity columns, and the 35-kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the p-subunit of the related Na+/K+-transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Nut1 Acad. Sci. 87, 6418-64221; (6) co-precipitated a molecule of 95 kDa with the 60 -90-kDa molecule from 2sI-labelled detergent extracts of dog tubulovesicular membranes; and (7) co-purified the catalytic a-subunit of the H '/K + ATPase with the 60 -90-kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6-Sepharose 4B column, indicating a physical association between the two molecules.These results provide further evidence that the 60 ~ 90-kDa glycoprotein is the p-subunit of the gastric H + / K + ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35-kDa core protein of the 60-90-kDa p-subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated p-subunit, as well as the ontogeny of the gastric proton pump.The parietal cells of the gastric mucosa are highly specialised for the secretion of hydrochloric acid into the stomach. Resting (non-secreting) parietal cells contain abundant smooth cytoplasmic vesicles called tubulovesicles. Upon stimulation by a variety of ligands e.g. gastrin, parietal cells undergo extensive morphological transformation resulting in hydrochloric acid secretion. These changes include an extensive reduction in the tubulovesicular membranes of the cytoplasm together with the appearance of intracellular secretory canaliculi [I, 21. Abbreviutions. H + / K + ATPase, H+/K+-transporting adenosine triphosphatase; Na+/K+ ATPase, Na+/K+-transporting adenosine triphosphatase; FITC, fluorescein isothiocyanate.Enzymes. H + / K + ATPase ( EC 3.6.1.3); Na+/K+ A...

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Western immunoblotting: Temperature-dependent reduction in background staining

Cited by 26 publications

References 14 publications

The stringent response, σH-dependent gene expression and sporulation in Bacillus subtilis

The stringent response, σH-dependent gene expression and sporulation in Bacillus subtilis

Immunocytochemistry and in situ hybridization in the electron microscope: combined application in the study of virus-infected cells

Monoclonal antibodies specific for the core protein of the β‐subunit of the gastric proton pump (H⁺/K⁺ ATPase)

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Western immunoblotting: Temperature-dependent reduction in background staining

Cited by 26 publications

References 14 publications

The stringent response, σH-dependent gene expression and sporulation in Bacillus subtilis

The stringent response, σH-dependent gene expression and sporulation in Bacillus subtilis

Immunocytochemistry and in situ hybridization in the electron microscope: combined application in the study of virus-infected cells

Monoclonal antibodies specific for the core protein of the β‐subunit of the gastric proton pump (H+/K+ ATPase)

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Product

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About

Monoclonal antibodies specific for the core protein of the β‐subunit of the gastric proton pump (H⁺/K⁺ ATPase)