Hello,
I would like to find the interacting partners of my protein of interest (POI) in tissue homogenates. The antibody specific to the protein of my interest is available. Since I would be using many samples ( and more experiments), I think, Immuno Affinity Chromatography (IAC) is better than Immunoprecipitation (IP), as it will be cheaper in the long run and also possible to get more reliable and reproducible results. Moreover, one could avoid some non-specific interacting partners of my POI. Having decided to follow IAC, I have the following questions to ask.
Questions:
1. Did anyone try IAC to screen for the interacting partners? If so, could you please suggest things that I should keep in mind during the method execution or planning.
2. What is the best method to couple the antibody to the column? Direct coupling using protein A or Protein G beads?
3. If I have to perform mass spec after IAC, what would be the expert's suggestion (related to performing the purification)?
Thank you all in advance!
It will be really helpful, if anyone could share the protocol to couple the antibody to the column.
I would not recommend to use IAC for screening of interacting partners. When you couple your Ab to any resin you couple it covalently in random way usually through Lys residues available NH2 groups. Therefore, the number of Ab molecules that will be in native conformation will be limited compared with their native reaction conformation. That's why the capacity of the affinity column will be much less than any capacity of the same Ab amount in solution. All above mentioned indicate to a better option to use IP for the screening of interaction partners.
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