After stimulation and incubation of our PBMCs for 24 hours we harvest 120ul of cell suspension (600.000 cells), centrifuge it, discard supernatant, and resuspend the cell pellet with 100ul mix of RIPA and PIC, cool 30 minutes on ice, centrifuge and aliquote supernatant.
As we aim to analyze phosphorylated proteins, e.g. IRAK4, we would like to adapt our extraction protocol. Is it sufficient to add phosphatase-inhibitors to the RIPA/PIC-mix? Which concentrations do you suggest?
Hi, if you're looking for the concentration of phosphatase inhibitors here is what I used as final concentration: 1 mM sodium pyrophosphate, 25 mM sodium beta-glycerophosphate and 50 mM of sodium fluoride and 1mM sodium orthovanadate.
Hello Alexandre,
In addition to Chaabouni's suggestion, several companies sell tablets containing mixtures of phosphoprotein phosphatase inhibitors. You just dissolve them in your RIPA (or make and store as a stock solution). Also make sure you include EDTA and EGTA to inhibit phosphates that require divalent cations for activity.
Thank you!
We use cOmplete EDTA-free Protease Inhibitor Cocktail. I'm not sure if I should add EDTA or just buy a new PIC.
I think PhosStop tablets by Sigma seem to be an appropriate and not too expensive tool to inhibit a broad range of phosphatases.
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