Blocked column

John Philo jphilo_nospam_ at mailway.com
Thu Apr 4 12:29:46 EST 2002


Alison,

Yes, resuspending your column material etc. is probably the best thing to
try.

What is not entirely clear is whether this blockage is due to contaminants
or to the protein you are trying to recover. With ion exchange the protein
can get so concentrated at the top of the column that aggregation is
induced, particularly when other contaminating proteins (or nucleic acid)
are present.

For that reason, and to do some preliminary removal of contaminants, it is
often a good idea to first do a batch-mode binding/release step (no column,
just in a beaker).  Then you can go to a column procedure, but you may still
want to do the column loading under pH/ionic strength conditions where the
binding of your protein is not so strong if your protein is intrinsically
prone to aggregating on the resin.

John Philo
Alliance Protein Laboratories

"Alison Richmond" <alison.richmond at strath.ac.uk> wrote in message
news:20020404171515.20264.qmail at ww02.jatek.com...
> Hi,
>
> I have just loaded on all my protein on to a DEAE-Sephacel column and the
> column now appears to be blocked, as some of the tubing has been forced
> apart due to back-pressure.  My protein is coloured and I can see it!! But
> not sure exactly how to recover it.  Should I be able to resuspend the
> column material, mix with a high salt buffer (which I  would normally use
to
> elute the protein) and then by centrifugation recover the protein in the
> supernatant, repeating the proceedure until the column matrix appears
white
> again?
>
> Any help and advice much appreciated,
>
> Alison
>
>
> <http://www.biowww.net/forum/read.php?f=6&i=988&t=988>
>
>





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