FPLC Column Cleaning Protocol?

[Tobias Straub]

In article <6p3png$ec0$1 at news.fas.harvard.edu> ,
wong5 at login2.fas.harvard.edu (Donny Wong) wrote:

>
>Dear netters,
>I've been having a  horrible time with a mono-S column on the FPLC...the
>back pressure is very high, causing the system to shut down very
>frequently.  I tried cleaning out the column with a protocol from
>Pharmacia (2M salt injection, 1N NaOH inject, 0.5% SDS/0.5 N NaOH), but
>the pressure is still quite high when i inject my sample and while washing
>the column afterwards.  I managed to inject my entire sample by stopping
>the machine every time the pressure got too high, waiting for it to go
>down, and then starting up the injection again (I did this for 25mls...and
>it shut down pretty much after every ml (at 0.1 ml/min).  finally i am
>washing and the pressure is stable, so i may be able to complete the
>purification.  so to make a long story short...is there a good protocol
>for cleaning this column after i'm done with it so that i (or anyone else
>in the lab) wont have to deal with this problem again?  I'm wary of
>Pharmacia's protocol...the 0.5% SDS/NaOH solution made the problem even
>worse when the SDS precipitated out of solution in the cold cabinet the
>system is in.
>
>thanks,
>donny wong
>wong5 at fas.harvard.edu

Seems to be more a mechanical problem. Try to check tubings and filters. A
protein/DNA etc.- plugged column should easily be cleaned by NaOH up to 2M,
HCl 1M and Urea (don't forget to run a normal buffer in between).

BTW: It's really hard to kill a Mono-column.


------------------------------------------------
Tobias Straub
Medizinische Poliklinik, University of Wuerzburg
Klinikstrasse 6-8
97072 Wuerzburg
Germany
Phone: +49-931-2017085
Tobias.Straub at mail.uni-wuerzburg.de