Pharmarcia Red Sepharose CL-6B

MESALV00 at ukcc.uky.edu MESALV00 at ukcc.uky.edu
Wed Feb 2 19:46:21 EST 1994


In article <sbbiovm.sunysb.edu-010294222758 at 129.49.19.31>
sbbiovm.sunysb.edu (Ing-Nang Wang) writes:
 
>
>I wonder if anyone has experience on Pharmacia Red Sepharose CL-6B?
>Recently, I tried to partially purify 6-phosphogluconate dehydrogenase (use
>NADP) from E. coli.  However, I found it very difficult to elute the enzyme
>from the column.  The elution buffer I used is HEPES 50 mM + MgCl2 10 mM +
>NADP from 1 to 3 mM.  Any idea on this?
 
Dear Ing-Nang Wang
 
Try incubating the column with the eluting ligand (i.e., NADP) for a longer
period of time before you elute the protein.  Alternatively, try using a
different dye resin, one that the dehydrogenase binds less tightly.  Generally,
dehydrogenases bind to orange, green and yellow resins less tightly than they
do to red or blue.  Finally, try eluting with both substrates.
 
Good luck,
 
Mike
 



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