I am planning to look at the influence of several proteins on insulin dependent phosphorylation of IR and IRS.
Hi Revathy,
The main concern in such experiment is to stimulate your cell at rest...
You have first to plate it or dilute it (depending if they are floating cells or adherent) at an optimum concentration the day before (usually I do this with floating B cells at 1Million/ml) to not spontaneously activate them...
And the day of the experiment you have to wash them at least twice with DMEM without FBS and without glucose to not activate phosphorylation. Using RPMI could affect your experiment cause it contains insulin.
After double washing, I split cells at the desired concentration into experimental eppendorphs tubes and let them rest in DMEM 0% no glucose no antibiotic for at least 1h.
In the case of my B cells I stimulate 1 million cell in 100 microliter; so I split them in 10million/ml and I put 100microL in a tube for 1h for resting.
I use DMEM for these cells that usually grow in RPMI 10% and my phosphorylation (here I evaluate Akt and p38) work as a storm.
Best luck.
Thanks Souhad,
do I have to immunoprecipitate before western blotting or I can proceed right away especially the insulin receptor beta and IRS both phosphorylated and non phosphorylated.
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