Sephacryl 500 column problem...

Szymon szymon at biotech.univ.gda.pl
Tue Oct 29 04:58:06 EST 2002


Hello everyone,

I'd like to ask you a little question about my problem. Tell me please,
what I am doing wrong.

I once wanted to separate some protein aggregates. I mean, big ones from
smaller and from monomeric protein. Through I've been strongly
discourtaged to do so, I took a bottle of Sephacryl HR 500 and poured a
1cm / 30 cm column according to Pharmacia documents. The first try I got
very small resolution (acetone run), second try it was  a bit too much
packed (leading peaks, as described in Pharmacia gel filtration booklet)
but third attempt was quite satisfactory for me.

I made some runs on it. My aggregates eluted somewhere in the middle of
elutin volume (they are not so very big after all). But recently the
back - pressure was increasing and the resolution droped very much. I
cleaned it with reccomended 0.2 M NaOH which improved a situation -but
only for a moment. Now I got some 0.1 - 0.15 MPa at the flow rate of 0.3
mL/min (previously, after packing, it was about 0.02 - 0.03). The buffer
I use is 10% glycerol, 150 mM salt and 20 mM TRIS pH 7.5 (yes, filtered
and degassed), it is running at about 6 centigrades. 

And when I run a mixture of Dextrane Blue 2000 with acetone, acetone
went out first, with quite broad peak with some tail, and Dextran is
still visible at the first 2 - 3 cm of column.
At last run, acetone eluted in 20%ethanol, the resin have even
compressed some 2 - 3 mm. It could not get stuck with acetone, could it?

Now I am quite puzzled - why the column is deteriorating so quickly? I'd
understood that there are some very big - or very sticky - aggregates,
but then why cleaning does not help? I thought about microbial growth,
but there is constant flow between experiments and for week-ends it is
filled with 20% etOH.
Other problem may possibly be some variation of temperature, as it is
kept in a kind of cooling cabinet, and recently a lot of people is to
open it allowing warm air inside (our cold room being rebuilded) :-(

Could anyone enlight me? I'd appreciate every suggestion,

Regards

 - Szymon Zietkiewicz
        (Ph.D student at Intercollegiate Faculty of Biotechnology, Univ.
Gdansk, Poland)

 szymon at biotech.univ.gda.pl



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