Hello everyone,
I would like to identify the protein(s) linked to a specific protein of interest which is secreted. I don't know if I can preform a co-IP directed to the protein of interest and then identify the potential protein(s) which is (are) linked to, by proteomic or other technique?
Thanks in advance :)
yes, you can do it! You can IP you protein of interest, then run a comassie gel and later go for Mass Spectrometry. I suggest to contact MasSpect facility in order to get the right protocols and reagents. Good luck
A word of caution, make sure to include appropriate -ve controls such as KO material. IP-MS experiments are prone to large number of false positives.
IPs for secreted proteins are always tricky because of their "sticky" nature. Be sure to use input material which is enriched for your protein of interest.
Good luck!
Comassie blue is not really so sensitive (50 ng) therefore I recommend you to stain your gel/gels with colloidal blue or silver staining (5-10 ng) which are more sensitive. If you have Typhoon or other gels fluorescence scanner then you could stain with Krypton fluorescence or sypro ruby staining ( is very sensitive 0.5 - 3 ng). All these staining methods are compatible to use for further protein identification with MS. One important thing is to have negative control such as NRS beside when you Ip you Protein X just to be sure that you don't pick up unspecific proteins.
Good Luck
I second Fuad's suggestion about silver staining if you cannot isolate enough protein to detect by CBB staining. You'll have to be careful when handling the gel throughout as the slightest protein contamination (usually by keratin) will affect the quality of your mass spectrometry analysis.
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