Protein Phosphorylation - (Aug/24/2006 )
Iam doing western blot for phosphorylated proteins in MAPK Signalling pathway.
Proteins within the cell are constantly phosphorylated and dephosphorylated, so the extent of
expression that we get in western blot, will it be consistent.
Whether the amount of phosphorylated protein will vary between experiments.
If so how will be able to get a consistent result?
I'm guessing you're looking at unstimulated cells plus cells that have been stimulated/treated in some way. If you stop the stimulation by removing the medium and putting on cold PBS and putting the flasks/plates on ice this should stop kinases phosphorylating proteins and your lysis buffers should have protease and phosphatase buffers in them to stop the proteins being dephosphorylated.
Hopefully you should see similar results when you repeat the experiment 3+ times.
Look at the material and methods papers of groups in your field for good methodology.
All the best,