PhastSystem/Western

Ron Tate rtate at bmb-fs1.biochem.okstate.edu
Fri Feb 16 10:08:05 EST 1996


John H Richburg wrote:
> 
> I am having trouble getting good protein separation using the PhastSytem
> so that I may ultimately do western blotting.
> 
> I suspect my trouble is in the sample preparation.
> 
> I homogenized my tissue with Dounce homogenizer in buffer containing 0.1 g
> CHES, 0.2 g SDS, o.1 g DTT, 1 ml glycerol in a volume of 10 ml (1.5 ml/100
> mg tissue).
> I then centrifuged for 2 h at 100,000 x g.
> My final protein conc. is 1 mg/ml.
> 
> To run on the gel. I did a TCA ppt and solubilized proteins in 0.1 N NaOH.
> I added 7.5 µl of sample to 2.5 ml sample buffer (50 mM Tris, 10%
> glycerol, 2% SDS, 0.1% BPB + mercaptoethanol).
> 
> I boiled. Then placed 4 µl of sample (=3 µg protein) on 20% homogeneous
> gel for separation using SDS buffer strips.
> 
> Coomasie stianed gel reveals a smear that is wide at the origin leading to
> a point at the bottom of the gel.
> 
> Could an experienced protein chemist/Phast user shed insight into my messy
> separation. Western blot of this gel gave only two non-specific bands near
> the origin.
> My proteins of interest are 26 and 40 Kd.
> 
> Help!
> 
> John
>John,
We use a Phast System in our lab so I've got a couple of ideas,
1)You don't need the glycerol in the sample buffer, are you using the 
sample combs or just applying 4 microliters directly on the gel?
2) 20% gel seems too high, I would think most of your proteins are 
staying near the top, even your 26 and 40KDa, I wouldn't expect them to 
be much resolved on a 20% gel.  We are interested in a ~36 KDa protein 
which runs near the middle of 10-15% gradient and 12.5% homogeneous 
gels.  For a crude sample I think I would try a 10-15% or 8-25% gradient 
gel.
3) I have done gels where I TCA precipitated dilute samples and 
reconstituted the pellet in buffer and mixed with sample buffer and they 
ran beautifully, yet at other times with the same kind of preparation it 
was not so good, I suspect the pellet needs to be rinsed maybe with 
acetone before reconstituting.

Not a lot of help but maybe some.
Happy Hunting  

-- 
Ron Tate                                                                
   
Lab of Franklin Leach
Dept. of Biochem. & Molecular Biology
Oklahoma State University 
e-mail rtate at bmb-fs1.biochem.okstate.edu



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