Peptide Competition and Phosphatase Treatment. Lysates prepared from HepG2 cells stimulated with TGFbeta were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-4) or treated with lambda phosphatase (5), blocked with a 5% BSA-TBST buffer overnight at 4°C, and incubated with 0.35 µg/ml Smad2 [pT8] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab and quote;)2 anti-rabbit IgG HRP-conjugate (Cat. no. ALI4404) and bands were detected using the Pierce SuperSignal™ method. The data show that the peptide corresponding to Smad2 [pT8] blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.
|Tested species reactivity||Human, Mouse, Rat|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human Smad2 that contains threonine 8. The sequence is conserved in mouse and rat.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 50% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||10µl|
|Flow Cytometry (Flow)||3-5 µg/million cells|
|Western Blot (WB)||Assay Dependent|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
SMAD2, also known as MADH2 or MAD2 regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation. Smad2 interacts with the TGF-beta receptors through its interaction with the SMAD anchor for receptor activation into the nucleus is a central event in TGF beta signaling. Phosphorylation of threonine 8 in the calmodulin-binding region of the MH1 domain by extracellular signalregulated kinase 1 (ERK1) enhances Smad2 transcriptional activity, which is negatively regulated by calmodulin.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.