Problems w/ native PAGE

[Paul A Bucciaglia]

Yasha at bioch.tamu.edu (Yasha Hartberg) writes:

>In article <bucc0003.783012079 at gold.tc.umn.edu>, bucc0003 at gold.tc.umn.edu
>(Paul A Bucciaglia) wrote:
>> I have been having problems with native PAGE of proteins.  When I extract 
>> total proteins from tobacco flower organs or leaves (I've listed my 
>> protocol below) and run them on 4 to 20% denaturing SDS gels, I get fine 
>> resolution of bands with no detectable smearing.  If I run the same 
>> amount (30 ug) on a 15% native gel, the bands are poorly resolved and 
>> smeared along the edges of each lane.  I have also noticed a band of 
>> precipitated proteins on the interface of the stacking and resolving 
>> gels; this occurs weather I layer the resolving gel with butanol OR water 
>> so I don't believe residual butanol is the main problem, as was mentioned 
>> earlier on the net. I also briefly centifuge the preps after adding 
>> loading buffer, so I don't believe that particulates are to blame.


>I have noticed that this problem is more pronounced in samples that contain
>both  BMe and EDTA, even more pronounced in samples with DTT and EDTA.  In
>addition, your concentration of BMe seems a bit high to me.  I would
>recommend playing around with your extraction buffer and see if the
>problems go away.

>Yasha Hartberg
>Texas A&M University


Hello Yasha,

After extraction with the EDTA and B-ME buffer, I ammonium sulfate ppt. 
the protein, rinse 1X in PBS, then resuspend in PBS and run over a spin 
column to desalt; do you think the B-ME and EDTA are causing denaturing or 
ppt. of the proteins during extraction?  What seems like a more 
reasonable B-ME concentration to you?

Thanks for the info!

paul bucciaglia