[Protein-analysis] Protein Crashing out of Solution

Kristina Blake kab69 at cornell.edu
Thu Apr 27 15:30:33 EST 2006

Hi All-
   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!
       Kristina Blake

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