Analysis Note

ControlPDGF treated NIH-3T3 cell lysates.

Application

ELISA:
A previous lot was recommended at 0.05 mg/mL by an independent laboratory for ELISA.

This Anti-Akt1 Antibody, clone 11E6 is validated for use in ELISA, WB for the detection of phospho-Akt1/PKBα (Ser473).

General description

The serine/threonine kinase Akt family contains several members, including Akt1 (also designated PKB or RacPK), Akt2 (also designated PKB-β or RacPK-β) and Akt3 (also designated PKB-γ or thyoma viral proto-oncogene 3), which exhibit sequence homology with the protein kinase A and C families and are encoded by the c-Akt proto-oncogene. All members of the Akt family have a Pleckstrin homology domain. Akt1 and Akt2 are activated by PDGF stimulation. This activation is dependent on PDGFR-β tyrosine residues 740 and 751, which bind the 85 kDa subunit of the phosphatidylinositol 3-kinase (PI 3-kinase) complex. Activation of Akt1 by insulin or insulin-growth factor-1(IGF-1) results in phosphorylation of both Thr 308 and Ser 473. Phosphorylation of both residues is important to generate a high level of Akt1 activity, and the phosphorylation of Thr 308 is not dependent on phosphorylation of Ser 473 in vivo. Thus, Akt proteins become phosphorylated and activated in insulin/IGF-1- stimulated cells by an upstream kinase(s). The activation of Akt1 and Akt2 is inhibited by the PI kinase inhibitor wortmannin. Taken together, this data strongly suggests that the protein signals downstream of the PI kinases.

Legal Information

UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Physical form

Purified mouse monoclonal IgG1κ lyophilized from 1 mL 2X PBS, PEG, sucrose, and 0.09% sodium azide.
Reconstitute with 1 mL H₂O (15 min. at RT). Concentration after reconstitution: 100 µg/mL

Format: Purified

Quality

Evaluated by western blot on lysates from mouse NIH-3T3 fibroblasts treated with PDGF.

Western Blot Analysis: 0.5-2 µg/mL of this antibody detected phosphorylated Akt1/PKBa in lysates from mouse NIH-3T3 fibroblasts treated with 100 ng/mL PDGF for 20 minutes.

Specificity

Recognizes phosphorylated Akt1/PKBα at Ser473, Mr 60 kDa. This lot of antibody does not cross-react with Akt1/PKBα dephosphorylated at Ser473.

Storage and Stability

Stable for 1 year at -20°C from date of receipt.
Reconstitute prior to use. Reconstitute with 1 mL H₂O for 15 min at room temperature. Reconstituted aliquots should be stored frozen at -80°C. Thawed aliquots may be stored at 2-8°C for up to 3 months. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.

Target description

60 kDa