Purification of FC tagged proteins - (Sep/27/2012 )
I am purifying a FC tagged protein on a Protein G column from pierce. I am getting a lot of protein expression, but when I load my protein on the column I am losing it all in the wash step and there is nothing in the elution.
Does anyone have any advice for me to not lose my protein in the wash step?
you can try slurry protein G instead of column. you need to bind anti-FC antibody to protein G by overnight incubation at 4C on a rotator. this method always works better for me than column. I can give you the protocol if you want.
If you could send me the protocol, that would be awesome! I've even tried decreasing what I load onto the column, but I still keep losing my protein in the wash.
Immunoprecipitation and Western blot
Twenty uL Protein G beads (KPL, USA) were washed twice with PBS and were then incubated with anti-Bax clone 6A7 (Zymed, USA) in a 0.5 mL microtube for 4 h to overnight at 4°C with end-to-end rotation. The beads were washed twice more with PBS and pelleted down for 3 min at 2400 rpm to remove unbound antibodies. The beads were then blocked with filtered 1% (w/v) BSA in PBS for 30 min. Later, normalized CHAPS-extracted proteins were incubated with the antibody-coated protein G beads overnight at 4°C on a rotator with gentle agitation. After this, the beads were washed three times with the solubilization buffer containing 0.5% (w/v) CHAPS buffer to remove all unbound proteins and were then pelleted down. Next, the beads were mixed with 20-50 ul SDS-PAGE sample loading buffer and boiled for 10 min at 95°C. In the final step, the beads were centrifuged at maximum speed for 10 min. The supernatant was subjected to SDS-PAGE and Western blotting with rabbit polyclonal anti-Bax N20 (Abcam, USA) or mouse monoclonal anti-Bax 2D2 antibodies (Zymed, USA). For all Western blot analysis 12.5% gels were prepared. High-speed centrifuge of the beads in all steps were avoided as speeds higher than 2400 rpm could rupture the beads.
@Smog187, you can use elution buffer instead of SDS-PAGE loading buffer .