Detection of Human and Mouse Phospho-Akt (S473) by Western Blot.
Western blot shows lysates of Jurkat human acute T cell leukemia cell line, MCF‑7 human breast cancer cell line, and NIH‑3T3 mouse embryonic fibroblast cell line untreated (-) or treated (+) with 100 nm Calyculin for 30 minutes, 1 µg/mL insulin for 5 minutes, or 100 ng/mL Recombinant Human PDGF-AB (Catalog # 222-AB) for 20 minutes. PVDF membrane was probed with 0.2 µg/mL of Mouse Anti-Human Phospho-Akt (S473) Pan Specific Monoclonal Antibody (Catalog # MAB887) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for Phospho-Akt (S473) at approximately 65 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Detection of Human Phospho-Akt (S473) by Simple WesternTM.
Simple Western lane view shows lysates of MCF‑7 human breast cancer cell line untreated (-) or treated (+) with 100 ng/mL Recombinant Human IGF‑I (Catalog # 291-G1) for 20 minutes, loaded at 0.2 mg/mL. A specific band was detected for Phospho-Akt (S473) at approximately 65 kDa (as indicated) using 2 µg/mL of Mouse Anti-Human/Mouse Phospho-Akt (S473) Pan Specific Monoclonal Antibody (Catalog # MAB887). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.
Preparation and Storage
Sterile PBS to a final concentration of 0.5 mg/mL.
Reconstitution Buffer Available
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
Stability & Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
6 months, -20 to -70 °C under sterile conditions after reconstitution.
Akt, also known as protein kinase B (PKB), is a central kinase in such diverse cellular processes as glucose uptake, cell cycle progression, and apoptosis. Three highly homologous members define the Akt family: Akt1 (PKB alpha ), Akt2 (PKB beta ), and Akt3 (PKB gamma ). All three Akts contain an amino-terminal pleckstrin homology domain, a central kinase domain, and a carboxyl-terminal regulatory domain. Akt1 is the most widely expressed family member and is frequently activated in a number of carcinomas, including breast, prostate, lung, pancreatic, liver, ovarian, and colorectal cancer. Akt1 is activated in a multistep process that involves the sequential phosphorylation of Thr450 by JNK kinases, Thr308 by PDK1, and Ser473 by PDK2 or mTORC2. Activated Akt1 phosphorylates a wide variety of cytosolic, nuclear, and mitochondrial substrates. Human Akt1 shares 98% aa sequence identity with mouse and rat Akt1.
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