Dear Proteomics experts,
I'm working on Arabidopsis to identify interacting proteins with plant immune receptor.
My immune receptor proteins is tagged with c-myc at its C-terminus and functions as wild type.
Myc-tagged proteins were IPed. There is a known binding partner. I confirmed Co-IP between immune receptor and its partner by WB with about 5% of final eluate.
For MS analysis, 95% of eluate was run in 1D SDS-PAGE. Once all samples separated from the top of resolving gel, the whole sample was excised and sent for MS.
During Co-IP, I used three different washing condition increasing salt concentration from 50, 150 and 300mM.
In the final peptide profiles, I could not get any peptides from bait (myc-tagged protein).
I guess that, if protein can be detected by WB, it could be expected to recover peptide from MS analysis although there are over 100 proteins detected from 50 and 150mM washing condition.
My myc-tagged protein is a membrane associated (does not have TM domain). I performed Co-IP with microsomal fractions from around 10g of plant tissue samples.
As a control exp., I also repeated Co-IP and ran SDS-PAGE fully. The bands around expected size of my bait proteins were also sent for MS analysis, but no result!!!
What can cause this problem? Can this problem be solved by scaling up the exp, with more samples such as 30 or 50g of tissues?
Thanks in advance for all your advice!
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