Western blot analysis of p70S6K (pTpS421/424) was performed by loading 30 ug of untreated HeLa cells (lane 1), HeLa cells treated with IGF1 (150 ng/mL for 15 minutes) (lane 2) and U-87 MG lysates (lane 3) using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), Xcell SureLock™ Electrophoresis system (Product # EI0002), Novex sharp Pre-stained Protein Standard (Product # LC5800). Proteins were transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour at room temperature. p70S6K (pTpS421/424) was detected at ~70 kDa using ABfinity™ p70S6K (pTpS421/424) recombinant rabbit monoclonal antibody (Product # 701083) at a 1:1000 dilution in 2.5% skim milk at 4° C overnight on a rocking platform. To confirm specificity, competition was performed with the phosphopeptide (10 ug/mL) (as shown in the corresponding blot on the right). Detection was performed using an HRP-conjugated goat anti-rabbit secondary antibody (Product # G21234) at a 1:5000 dilution and chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
|Tested species reactivity||Human, Rat|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Phosphopeptide corresponding to amino acids 417-428 of human p70 ribosomal protein S6 kinase|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||1 ul|
|Flow Cytometry (Flow)||1:100-1:500|
|Western Blot (WB)||1:500-1:5000|
* 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.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale, and purified with Protein A.
ABfinity™ monoclonal antibodies resemble rabbit monoclonals isolated from serum or produced by hybridomas, but demonstrate greater specificity and sensitivity. Because ABfinity™ recombinant antibodies are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
p70 Ribosomal Protein S6 Kinase (p70S6K) is a 70 kDa member of the ribosomal S6 kinase (RSK) family of serine/threonine kinases. It is predominantly localized in the cytoplasm, and is essential in growth factors regulated cell proliferation, pathways involving cell motility, such as metastases, the immune response, and tissue repair. p70S6K acts downstream of phosphoinositide (PI) 3-kinase, and its main physiological target is the S6 ribosomal protein, which is involved in upregulation of protein synthesis.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.