1) What is AKT?

The serine/threonine kinase B AKT pathway (also known as the PI3K-Akt pathway) plays a vital role in the regulation of cellular processes, including cell proliferation, survival, and growth – processes that are essential for oncogenesis. Mutation of the regulator proteins PI3K and PTEN causes uncontrolled disruption within the PI3-kinase pathway, leading to the development of human cancers (1,2; see also AKT pathway poster for more details).

2) phospho-AKT and FAQs

A) What is the best way to normalize phosphorylated proteins analyzed by western blot?Normalize phospho-AKT and total AKT with your loading control (e.g. Actin, tubulin), then calculate the phospho/total ratio using these normalized values. Put more simply:1. Calculate the ratio of band intensities of a phospho-AKT band: the loading control.2. Calculate the ratio of band intensities of total AKT: loading control.3. Divide ratio obtained #1 by #2 to obtain a normalized value for comparison among different conditions. This procedure allows one to distinguish between a change in AKT expression and a change in the ratio of phospho-AKT.* If you are looking at the differences in a phospho-AKT expression resulting from an experimental condition (e.g., knockdown), you should also show the expression of total AKT to distinguish between a change in AKT expression (transcription/translation level) and a change in the AKT phosphorylation status.B) What is the observed molecular weight for AKT and phospho-AKT?Molecular Weight AKT – 56 kDaMolecular Weight phospho-AKT – 60 kDa (Figure 1)

Figure 1. WB: HEK-293 cell lysate was subjected to SDS PAGE followed by western blot with 60203-2-Ig (AKT antibody) and 66444-1-Ig (AKT-phospho-S473 antibody) at a dilution of 1:4000 incubated at room temperature for 1.5 hours.

C) Are there any special WB conditions to optimize staining of a phospho-AKT?Since this is a phosphorylated protein, 5% BSA is recommended over non-fat milk as a blocking agent.D) What are good positive and negative controls for a phospho-AKT?- Positive Control: HEK293 cells- Negative Control: Treatment with PI3K inhibitors (e.g. wortmannin)E) What species does this antibody react with?

Our internal testing has confirmed that it reacts with the human and mouse forms of phospho-AKT.Reactivity with the human form is also supported by the literature’s citations of this antibody.

References:

1. Perturbations of the AKT signaling pathway in human cancer.2. Targeting the PI3K-Akt pathway in human cancer: rationale and promise.