Concentrating proteins already in SDS sample buffer?

brett brett at BORCIM.WUSTL.EDU
Fri Aug 30 15:29:59 EST 1996


>Hi all.
>
>Exactly what the subject says.  I'm looking for a way of concentrating a 
>protein sample that I have already treated with SDS_sample buffer.  The 
>sample is pretty high in salts already so I don't want to simply freeze 
>dry it.  Does anyone have any sort of precipitant method that could be 
>used - would cold MeOH work.  I want to concentrate the sample so that I 
>can get enough of a fairly minor band onto pvdf for N-terminal sequencing.
>
>Answers prefered via email.
>
>Thanks,
>Yak

I use this method for quantitative protein recovery even from dilute solutions,
with or without detergents (from 0.1ml but can scale to need):
        0.4ml MeOH (at RT)
        vortex, pulse-spin down
        0.1 CHCl3
        vortex, pulse-spin down
        0.3 H2O
        vortex. Spin hard 1-5'
        Carefully remove upper phase
        ppt. proteins with 0.3ml MeOH
        vortex. Spin hard 2'
        remove sup and dry pellet.
The reference is Anal. Bioch. 138:141-143. I have found this to be *much*
better than TCA or acetone precipitation. For very dilute solutions I have
added carrier proteins to ensure absolute recovery.


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




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