Hi, I am now detecting the phosphoproteins (p-Ser) of mammalian cancer cell. However, these proteins can easily be de-phosphorylated so I try to lyse the cells with SDS dye immediately after washing with PBS. But this may cause the inaccuracy of housekeeping protein, and I need to do the western blot once more after normalisation. If I lyse the cell with RIPA buffer, what kind of phosphatase inhibitors and what are the concentrations of them required? In addition, will sonication lead to the de-phosphorylation of proteins after RIPA lysis? Thanks in advance.
I homogenization my samples in MPER buffer from Thermo Scientific and use Halt Protease and Phosphatase inhibitor cocktail from Thermo Scientific. The inhibitors and EDTA (both included in the kit) are added in a 1:100 dilution. I have used a polytron and sonicator to process my samples with the above buffer and inhibitor and had no issues. The key to protecting phosphorylation sites in my opinion is the following.
1. always keep your samples cold
2. avoid freeze thaws of your sample
3. when performing westerns, block in 2-5% milk but incubate the primary in 2-5% BSA
4. use HRP substrate and not alkaline phosphatase substrate to visualize your proteins.
Hello Gongli Cai
RIPA lysis buffer is useful for whole cell extracts and membrane-bound proteins, and also preferable for extracting nuclear proteins. It will disrupt protein-protein interactions.
The composition of RIPA lysis buffer is as follows:
150 mM sodium chloride
1.0% NP-40
0.5% sodium deoxycholate
0.1% SDS (sodium dodecyl sulfate)
50 mM Tris HCL, pH 8.0
You can add Sodium Orthovanadate which is an inhibitor for protein phosphotyrosyl phosphatases at 1mM final concentration and Sodium Fluoride which is an inhibitor for protein phosphoseryl and phosphothreonyl phosphatases at 5mM final concentration. Both these phosphatase inhibitors must be added fresh to the RIPA lysis buffer when you perform cell lysis. You should also add protease inhibitor cocktail fresh to the RIPA lysis buffer. Please perform cell lysis on ice.
I would not recommend sonication as it generates lot of heat and is not good for many proteins. Ice cold RIPA lysis buffer should suffice.
I hope this helps.
Good Luck.