affinity purification and proteomics - (Oct/01/2009 )
I have proteins that have been selectively labeled with a biotin moiety in a crude lysate. These biotinylated proteins represent about 5% of the total protein present. I want to pull down these proteins using streptavidin agarose, then do a trypsin digestion on resin, and analyze then analyze the results peptides by LC-MS/MS to identfy the proteins I pulled down. Here is my problem....I have a large volume of lysate (~ 4 mL) due to protein solubility issues. I am wondering what sort of set up I can use to expose the lysate to the streptavidin beads and be able to recover the solution from the beads. Does anyone have any suggestions?
Syringe barrel with frit (eg. empty SPE column) and stopcock?