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affinity purification and proteomics


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#1 Trying-2-Graduate

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Posted 01 October 2009 - 09:00 AM

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?

#2 K.B.

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Posted 01 October 2009 - 11:05 AM

Syringe barrel with frit (eg. empty SPE column) and stopcock?




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