I am following standard protein precipitation. Mix 1:1 sample and 50% TCA for a final TCA of 25%. Ice 10 min then centrifuge 14K RPM 4 degrees 5 min and I see a lovely translucent pellet (30ug proteins per pellet). Then I proceed to wash twice with acetone and now, following centrifugation as above, I see no pellet. Is this normal? Will i have anything on my SDS PAGE?
Typically, I've always seen a pellet post-acetone wash, but the only way to truly tell if you still have protein is to run it on a gel! Also, be very careful when removing the acetone supernatant because the pellets that I have observed are very light/don't often remain stuck to the side as I extract the acetone. Good luck!
I used TCA for protein precipitation and I washed the precipitated protein with acetone and I always have a precipitated protein.
try to use filtration instead of centrifugation to recover your protein after acetone addition .
Hi,
That's exactly what happened to me! I think maybe the ability of acetone for protein precipitation is less than TCA.
I saw a pellet in 20%TCA, but after the washing with acetone, all pellets disappeared! Then, I tried to do this with more protein, I mean that I raised the volume of the sample (from a 1.5 ml vial to a 15 ml falcon), and the pellet observed (not as much as precipitation after TCA adding).
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