Gel filtration in urea or Gu-HCl?

[Ding Ming]

>Column specs:
>
>80x1cm
>flow rate=5ml/hr
>fraction size=1ml
>
>Helmut
>
>
Dear Helmut,

As I understand so far, Sephacryl S-200 should be packed in a 
special way to get good resolution. Since you used 80X1cm column
here rather than the standard XK 16/70 etc., I assume you didn't use
prepacked column, therefore, how to pack the column could be the 
first question you should ask yourself. Follow Pharmacia's 
instruction.

Secondly, the flow rate should be monitored closely. For a standard 
XK16/70, I used to use 50 to 75 ml per hr. It seems that your flow 
rate is a little bit smaller than it should be.

For the experiment using detergent buffer, it is important to 
equilibrate the column with SUFFICIENT volumes of starting buffer. 
It would be nice if you can incubate your standard protein sample in 
starting buffer for a while to make sure the denaturation and it may 
not be neccesary in case of these standards are labile to the 
denaturants.

Good luck!



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    Ding Ming                     
    Telephone:(608)265-3544/Fax:(608)262-7420  
    Internet: ming at calshp.cals.wisc.edu/ming at zeus.ahabs.wisc.edu
    Sweet Protein Section           
    Taste Research Lab               
    Department of Animal Health & Biomedical Sciences    
    University of Wisconsin-Madison, 1655 Linden Dr., Madison, WI53706      
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