Question 1: After reconstituting the lyophilized protein with water, what is the composition of the buffer the cofilin protein is in?
Answer 1: The protein should be reconstituted to 5 mg/ml by the addition of 20 µ, l of distilled water. The protein will be in the following buffer, 10 mM Tris pH 8.0, 1 mM EGTA pH 8.0, 5% sucrose, and 1% dextran. In order to maintain high biological activity of the protein, it is strongly recommended that the protein solution be supplemented with DTT to 1 mM final concentration
Question 2: To separate the F-actin bound to cofilin, is it absolutely necessary to spin the sample at 100, 000 x g?
Answer 2: If possible we recommend spinning the F-actin/cofilin complex at 100, 000 x g to absolutely insure that the protein complex pellets. Check with your university&rsquo, s core facilities or neighboring labs for access to an ultracentrifuge. However, in a pinch, spinning at 50, 000 x g should pellet the majority of the complex.
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