2-D gels with phosphoproteins

Enter Your Name Here user at host.uci.edu
Thu Oct 19 00:31:48 EST 1995


In article <prenard-1710951145180001 at mickeymouse.biocell.fundp.ac.be>,
prenard at biocell.fundp.ac.be (Patsy RENARD) wrote:

> Is there anyone who has already been successfull wit bi-dimensional gels
> run on with 32P-labelled proteins ? If so, HOW ?
> 
> I am using the Pharmacia Biotech system with precast immobiline dry strip
> gels for the first dimension and precast SDS polyacrylamide gradient gels
> for the second dimension. I get very good results when I stain the gels
> with silver. But if I run on phospho-labelled proteins (derived from
> classical in vivo labelling protocols), I get a important background on
> the autoradiography, making any analysis of the gel impossible.
> Could you please indicate me how to decrease this background ?
> Thanks for your attention.
> 
> Patsy
> 

Dear Patsy

The problem you have is partly due to the presence of nucleic acids. In
order to get git of the DNA/RNA, you should either treat your samples with
DNase and RNase, or centrifuge at 200,000 x g for 1 hour your samples.
This way, you should be able to decrease the background. However, as 32P
is a strong isotope and almost all proteins are phosphorylated, do not
expect to get clean patterns. Your are not the first one to try and,
personally I think that's impossible. Good luck!
Feel free to contact me for additional advices.

Email: dmthomas at uci.edu 

Didier.



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