Hi All,
I was wondering if anyone had used the chormotek gfp trap magenetic agarose bead kit for purification of GFP tagged nuclear proteins.
I am attempting to purify a gfp-tagged protein (~230kD) from transiently transfected HEK293T cells (transfection efficiency is high and the cells are blazing). I have had some issues with large clumps of unlysed cells/DNA after adding RIPA buffer (add more DNase or too many cells?).
Has anyone performed this protocol for nuclear proteins and have any additions to the chromotek protocol which were helpful?
Thanks,
Aidan
I used their protocol, and it worked pretty good, just check the compatibility of your buffer with the beads. However, I had a lower yield that the claimed one (4 mg of protein/ mL of resin).
I also had similar issues when I used a large cell pellet. Most likely the clumps is DNA and it helps to add more DNAse and MgCl2. I also mixed during cell lysis several times with a pipette. You can also use a larger volume of RIPA buffer than indicated in the manual, in my case it still worked well with 500 µL RIPA buffer.
Maybe you should also check their GFP-Trap Dynabeads. I think they recommend these for very large proteins.
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