Sephacryl 500 column problem...

dans_green at hotmail.com dans_green at hotmail.com
Tue Oct 29 15:16:49 EST 2002


We regularly work with s200, and large s200 at that. I would look immediatley at growth. Our last column started acting wierd and after much exploration we found growth in 0.05% Sodium Azide! My suggestion would be to unpack the column. Place it in distilled h20 and autoclave it at 250 F, 18 psi, for 20 minutes. Then wash with 0.2 M NaOH. The NaOH will clean the endotoxin off the beads. The column can be clear and still have growth. Bacteria are resilliant little suckers.

Also, when you packed your column how much time did you give it to settle before you started running markers? The column will slightly pack over time.

I would not park the column in 20% etOH. Use Sodium Azide. Most people recommend 0.05%. Make sure that all of your pipettes are sterile, that you run all samples and markers through a filter before adding them to the column. Also make sure that all of your reagents are either filter our autoclave sterile. Also sterilize all the fittings and tubing that you use. Some bacteria are very motile and will swim up the column. 

Finally, you should test the column every week for growth. We use blood agar plates. You can even test a sample of sodium azide. Good luck, I understand your frustration.

Daniel

Szymon wrote:

> 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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