I am currently working on the identification of phosphorylation on a specific protein, labeled as 'X,' from cell lysates. My approach involves immunoprecipitation using antibody 'X.' While this antibody performs effectively in immunoblotting, successfully pulling down protein 'X,' I have encountered challenges in its detection during IP-Mass spec analysis.
For cell lysis, I am utilizing RIPA buffer and implementing on-bead digestion followed by mass spectrometry. I am reaching out to seek any insights or suggestions from fellow researchers regarding potential improvements in the pull-down technique, overall protocol, or any recommendations to enhance the success of this procedure. Your valuable input would be greatly appreciated.
HI,
your antibody could be innefective in recognizing your protein by IP; the epitope can be masked by other interacting protein or be accessible only on denatured protein (western blot...), try another antibody, try to put a tag on your protein...
As Didier Poncet suggested, you might try a Western blot or ELISA to confirm your Ab pulls down your target, although as Didier Poncet hinted, sometimes Ab work well in one system and poorly another (i.e., IP vs Western vs ELISA).
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