SpecificityThis antibody does not cross react with RPS6 phosphorylated on serines 244 and 247.
Tested applicationsWB, ICC/IF, Electron Microscopymore details
Human Predicted to work with:
Synthetic peptide derived from the region of human RPS6 that contains serines 235 and 236 (unfortunately, the amino acid sequence is considered to be commercially sensitive)
HeLa +/- TNF-alpha or anisomycin
General notesCentrifuge before opening to settle contents.
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
Storage bufferPreservative: 0.05% Sodium Azide
Constituents: Dulbecco's PBS without magnesium and calcium, 50% Glycerol, PBS, 1mg/ml BSA, pH 7.3
Concentration information loading...
PurityImmunogen affinity purified
Purification notesPurified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated RPS6. The final product is generated by affinity chromatography using a RPS6-derived peptide that is phosphorylated at serines 235 and 236.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000. Detects a band of approximately 32 kDa (predicted molecular weight: 31 kDa).
Use at an assay dependent concentration. Used at a dilution of 1/100 for 2 hrs on HeLa cells (see Abreview submitted by Marco Biggiogera).
Use at an assay dependent dilution.
FunctionMay play an important role in controlling cell growth and proliferation through the selective translation of particular classes of mRNA.
Sequence similaritiesBelongs to the ribosomal protein S6e family.
Post-translational modificationsRibosomal protein S6 is the major substrate of protein kinases in eukaryote ribosomes. The phosphorylation is stimulated by growth factors, tumor promoting agents, and mitogens. It is dephosphorylated at growth arrest.
Western blot - RPS6 (phospho S235 + S236) antibody (ab12864)
Predicted band size : 31 kDa
Lysates prepared from HeLa cells left untreated (1) or treated with anisomycin (2-5) were resolved by SDS-PAGE on a 14% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with ab12864 for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with a goat anti-rabbit IgG HRP conjugate secondary antibody and bands were detected using the Pierce SuperSignalTM method. The data show that only the peptide corresponding to RPS6 blocks the signal, verifying the specificity of the antibody.
Immunocytochemistry/ Immunofluorescence - RPS6 (phospho S235 + S236) antibody (ab12864)This image is courtesy of an Abreview submitted by Dr Marco Biggiogera
ab12864 at a 1/100 dilution staining human HeLa cells by ICC/IF. The cells were formaldehyde fixed and then incubated with the antibody for 2 hours. An Alexa Fluor ® 488 conjugated goat polyclonal antibody was used as the secondary.