I want to check the phospho-AKT level in the lysates of Bone-marrow derived macrophage infected with different kind of viruses. So, i am looking for a good positive and a negative control to run on the sides for my reference.
Akt protein is downstream protein of insulin and also amino acid signaling pathway, so to get a strong band for Akt phosphorylation (Phospho-Akt), you can use protein from cell that had been induced by insulin as positive control. I assumed that imunological cell such as limphocyte and macrophage have insulin receptor in their membran and can be stimulated by insulin as well.
I agree with Elizabeth - stimulation with a growth factor (insulin especially, if the receptor is expressed) will induce Akt phosphorylation. Starve the cells of serum and growth factors first for a few hours or overnight (depending on what your cells can tolerate) and include lysates from stimulated and unstimulated cells. You should see a nice induction of phosphorylated Akt on Ser473 (also T308) following stimulation. You could also try inhibiting PI3K in your cells, to reduce the baseline level of Akt phosphorylation. This would be an additional negative control. Wortmannin (typically 0.1-1 micromolar) or LY294002 (typically 10-50 micromolar) are two commonly used PI3K inhibitors.
Treating cells with platelet derived growth factors (PDGF), induces AKT phosphorylation (Ser 473). In contrary, If you treat your cells with PI3 kinase inhibitors such as Wortmannin or LY294002, it reduces AKT phosphorylation substantially. Hope it helps.
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