I wanted to analyze the phosphorylation of a certain mammalian protein. It is a standard procedure to add phosphatase inhibitors to extraction buffers to block the process of dephopshorylation. Is it recquired to block the kinases during the process? If yes, what are the commonly used chemicals to do so?
I am assuming that you want to measure the phosporylation status of your protein by Western Blot. You could use RIPA lysis buffer with protease inhibitor tablet from Sigma. We have had success measuring phosphorylation of a wide variety of signaling proteins when the cells were lysed in Laemmli buffer.
Kinases are enzymes that phosphorylate proteins. So whether or not to add kinase inhibitors to your cells would depend on whether or not you want to prevent your protein from getting prosphorylated. If you want to see phosphorylation, you should not add kinase inhibitors. If you want to inhibit phosphorylation of your protein, pre-treat your cells with the relevant kinase inhibitor before adding your stimulants (30 min-1 hour pre-treatment is sufficient) and culture your cells in the presence of the inhibitor till you harvest the cells for WB. It is better to use specific kinase inhibitors than ones that can inhibit multiple kinases (it might be hard to interpret the data when multiple signaling pathways are inhibited and it might also affect cell survival). Let me know if you have trouble finding one.
There are plenty of broad spectrum kinase inhibitors: https://www.tocris.com/pharmacology/broad-spectrum-protein-kinase-inhibitors
For sample preparation for WB we also use protease inhibitor tablets (as Julie Joseph recommended).
Staurosporin is a good broad spectrum kinase inhibitor. Although I would not recommend using kinase inhibitors if you are going to look at protein phosphorylation. You can add a cocktail of protease and phosphatase inhibitors in your lysis buffer. Check this:
http://www.merckmillipore.com/IN/en/product/Calbiochem-Protease-and-Phosphatase-Inhibitor-Cocktails,MM_NF-C134544
Hi Dawid,
Short answer is: No! You do not need to add Kinase Inhibitors to your Lysis buffer!
The in vitro phosphorylation (or the lack of it) you want to measure is already there - it won't increase "ex vitro" upon lysis. And to keep what you got you add the aforementioned protease/phosphatase inhibitors.
Hope that helps!
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