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Synthetic peptide conjugated to KLH derived from within residues 250 - 350 of Human Pyruvate Dehydrogenase E1-alpha subunit, phosphorylated at S293.
Our Abpromise guarantee covers the use of ab92696 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 43 kDa (predicted molecular weight: 43 kDa).|
|ICC/IF||Use a concentration of 1 µg/ml.|
|IHC-P||Use a concentration of 1 µg/ml.|
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour and then treated with either buffer (lane 1) or alkaline phosphatase (lane 2), before being incubated with ab92696 overnight at 4°C. Antibody binding was detected using IR-labelled goat anti-rabbit (green) and goat anti-mouse (Red) at 1:10,000 dilution for one hour at room temperature before imaging.
ICC/IF image of ab92696 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab92696, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) HeLa cells at 1µg/ml.
IHC image of Pyruvate Dehydrogenase E1-alpha subunit (phospho S293) staining in Human lung adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab92696, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
This image is courtesy of an anonymous abreview.
Serially diluted ab92696 was bound to immobilised Phospho peptide (S293) - or Control peptide (1 microgram x mL-1). The antibody was detected by HRP-labelled goat anti-rabbit IgG
ab92696 staining Pyruvate Dehydrogenase E1-alpha subunit (phospho S293) in Human HUVEC cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100 pH 7.4 for 5 minutes and blocked with 5% BSA for 20 minutes at room temperature. Samples were incubated with primary antibody (1/500 in PBS) for 1 hour . A CF488-conjugated Donkey anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.
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