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Peptide Competition and Phosphatase Stripping. Extracts of CHO-T cells transiently transfected with wild-type human IRS-1 and treated with 100 ng/mL TPA for 30 minutes were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4°C and either left untreated (2-6) or treated with lambda phosphatase (1), then incubated with the IRS-1 [pS312] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1-3), the non-phosphopeptide corresponding to the phosphopeptide immunogen (4), a generic phosphoserine-containing peptide (5), or the phosphopeptide immunogen (6). After washing, the membrane was incubated with goat F(ab')2 anti-rabbit IgG alkaline phosphatase (Prod # ALI4405) and signals were detected using the Tropix WesternStar™ method. The data show that only the phosphopeptide corresponding to IRS-1 [pS312] blocks the antibody signal, 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|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human IRS-1 that contains serine 312. The sequence is conserved in mouse, rat and pig.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|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.
Insulin receptor substrates (IRS) are responsible for several insulin related activities, such as glucose homeostasis, cell growth, cell transformation, apoptosis and insulin signal transduction. Serine/threonine phosphorylation of IRS-1 has been demonstrated to be a negative regulator of insulin signaling and is responsible for its degradation, although IRS-1 degradation pathways are not well understood. IRS-1 has also been shown to be constitutively activated in cancers such as breast cancer, Wilm and quote;s tumors, and adrenal cortical carcinomas, thus making IRS-1 phosphorylation and subsequent degradation an attractive therapeutic option. To date there have been four subtypes identified: IRS-1,2,3, and 4, with IRS-1 being widely expressed.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Insulin receptor substrate 1; Insulin receptor substrate-1; IRS-1; IRS1