Hi ,
I am using invitrogen protein G dynabeads to enrich proteins, but the yield I am getting is around 20 ug. I need to do CID MS after pull downs, and require atleast 100ug proteins in initial elute so, that it will be sufficient enough for MS after tryptic digest, alkylation and cleanup steps.
Is there any other ways to get more yield in the final elute ? Currently I am loading 1 mg of whole cell lysates and incubating it overnight with antibody-bead complex.
The yield will be of course higher as you scale up the prep. You might look to optimize the concentration of your sample going on the beads to make sure you have maximized binding for the weight of bead you are using. But, if you're sample limited at this point, I'd focus on finding an alternative mass spectrometry resource. Most modern proteomic instrumentation can work just fine at that scale - 20 ug captured protein to digest and analyze.
Thanks a lot Andrew K Ottens .
The problem I am getting is that the final sample after digestion and HLB cleanup using C18 column is immeasurable by QUBIT; as QUBIT show it below its range. When I ran those samples on MS, I get 200 proteins and around 1000 peptides, but not all of them are specific to my antibody. I might be loosing some of my peptides during C18 cleanup.
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