concentrating protein lysates

Stephen R. Lasky, Ph.D. Stephen_Lasky at brown.edu
Tue Dec 13 11:14:21 EST 1994


In article <D0r5yM.4uz at ncifcrf.gov>, reming at ncifcrf.gov (Mary Remington) wrote:

>  We have isolated cellular protein from mammalian cells.  This is total
>  cellular protein we are using in a western blot.  We are having a 
>  difficult time seeing the protein of interest with our polyclonal Ab.
>  Is there a way to concentrate total cellular protein?  Thanks, Mary

Since you don't need active protein, there are several methods.  

I have found making an acetone powder the easiest:  Just add 3 volumes of
ice cold acetone to your protein suspension and let it sit overnight at
-20 deg C.  Spin down the ppt, dry and resususpend in a small volume of 1
X laemli buffer and run on the gel..

Other people use 10% TCA ppt (similar to above but using TCA), but I have
found that pH changes in this method can sometimes cause problems when
resuspending the proteins.

You could use vacuum dialysis (that could even give you an an active
product) if you have the aparatus.  Or in the same vein, you could use lmw
cutoff centricon filters to concentrate the protein.

You could also put your protein suspension in a dialysis tube, close both
ends and cover it with dry PEG.  That suck the water out pretty quickly.

These are all pretty simple techniques.  Of course, you could partially
purify the protein and in that way avoid the possibility of overloading
your gel.  Good luck

SRLasky

-- 
*********************************************************************
Stephen R. Lasky, Ph.D.
Roger Williams Medical Center/Brown University
Phone: 401-456-6572       Fax: 401-456-6569       e-mail: Stephen_Lasky at brown.edu
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"To me at least, 'Yuck' doesn't capture the full essence of death by
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