I want to do western blot for phosphorylated serine/threonine kinase protein. How can I prevent the target phosphorylated protein from dephosphorylation during protein extraction and doing western blot? What are the things I need to be careful about during western blot? What is best for blocking the membrane while doing western blot for phosphorylated protein?
You should add a phosphatase inhibitor to your extraction buffer. A standard ones are NaF, and Na orthovanadate.
Dear Vasily Ogryzko, thank you very much for your suggestion. Which blocking solution should I use? Milk or BAS or anything else? What would be their percentage in blocking solution? Thanks.
Thermo Scientific has a cocktail that inhibits different phosphatases, it is very good. I usually use BSA 5% for blocking membranes, but some antibodies specify in the datasheet the conditions you should use.
Hi Atiqur,
avoid milk powder for the blotting in phosphoproteomics - casein is often used as a standard to test methods for the detection of protein phosphorylation, as it is phosphorylated. It is actually quite tricky to get good Western Blots where you get an accurate idea of the general state of phosphorylation of a biological system. A good few of the problems described in this paper are still around:
Grønborg M, Kristiansen TZ, Stensballe A, Andersen JS, Ohara O, Mann M, Jensen ON, Pandey A.: Mol Cell Proteomics. 2002 Jul;1(7):517-27.
A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate.
You might also want to consider a peptide-centric mass spectrometry-based approach to find your phosphorylation sites, if that was at all available to you. If you start reading 'similar articles' to the one highlighted above in pubmed, you will quickly get an idea of what has changed in a dozen years.
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