IUBio

N-acetyl transferase purification, What to do next?

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Tue Mar 9 10:39:16 EST 1999


In article <36E02370.324E5276 at hotmail.com>, Biology <mpiraee at hotmail.com> 
wrote:
>Hi evryone
>It is a long time I am trying to purify N-acetyl transferase from
>Streptomyces venezuelae. It works on a specific substrate p-nitro phenyl
>serinol. I fractionated the crude cell extract by 60% amonium sulfate
>(after removing the pellet fro 30% am. sulfate) which is highly active.
>Then on DEAE collumn it was again fractionated by NaCl gradient. I found
>the active fraction with the highest activity. I analyzed that fraction
>with HPLC. It showed still it has two more proteins with very close MW.
>I already tried HIC columns but it did not worked. I need the peptide to
>be sequenced
>Any commnet on how to proceed for further purification is welcomed.
>

It is a standard practice to do higher resolution ion exchange
after initial crude purification. If you have an access to FPLC
or HPLC, do chromatography on Mono Q - it's a different 
chemistry and has very high resolution. It is likely to help.

Alternatively, try hydroxyl apatite chromatography. (I'd
recommend ceramic HA from Bio-Rad). There is no reason
high resolution HIC should not work too. Just play with
conditions... Finally, high resolution chromatofocusing 
(FPLC/MonoP) virtually gurantees you a nice separation
if you are lucky and your ptotein does not precipitate
during it.

Hope this helps,

        Dima



More information about the Proteins mailing list

Send comments to us at biosci-help [At] net.bio.net