[Protein-analysis] Re: Protein Crashing out of Solution

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Tue May 2 07:47:33 EST 2006


Kristina Blake wrote:

> 
> 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.

Do you have any detergent in your isolation buffer? If you go from high
to low detergent, or from one detergent to another, precipitation may
occur. 

Also, if you have a negatively charged detergent like SDS in your
isolation buffer and add bivalent cations like Mg, the detergent will
precipitate and may or may not take the protein down as well. 

Check wether Brij is actually dialysable
(http://psyche.uthct.edu/shaun/SBlack/detergnt.html), I am currently
offline. Also check that your 0.35% are above the cmc.

Could the pH of your PIPES buffer be close to the pI of your protein
(isoelectric focussing will tell you)? That too would reduce the
solubility of your protein.  

Mg might precipitate proteins, although that's rare.

The problem is that you are changing several factors at once, so it is
difficult to pinpoint the problem. Do small size experiments where you
only change one factor (pH, buffering ion, detergent, bivalent metal). 


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