I am trying to purify a protein from Insect Cells (SF9) and
everything works perfectly, clean protein, good yield etc., until I do a
dialysis step into the actual buffer I need to use for my experiments.
The purification/elution buffers are all 20 mM Tris plus 100 mM KCl
pH8.0 and then I need to switch to a PIPES buffer 100 mM KCl, 2 mM
EGTA, 1 mM MgSO4, and Brij-35 0.25% pH6.8. During this overnight
dialysis is when the protein crashes, when I take the dialysis cassette
out the next day I can actaully see "blobs" of protein!
I need to switch into the PIPES buffer since I will be doing
protein/microtubule assays and microtubules "like" the PIPES buffer.
Also I've never seen the assays performed with more then 100mM salt or
more then 0.25% detergent presummably becasue people feel this would
disrupt the microtubules.
Anyone have any suggestions? I tried upping the pH of the PIPES
buffer to 7.5 but that had no effect. Also I don't think the salt plays
that big of a role, because in my purification at one point (during a
TEV cleavage step) there is no salt present only TRIS and EDTA.
Thanks for any suggestions!