FPLC Column Cleaning Protocol?
Donny Wong
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
wo...@fas.harvard.edu
PELBIOP
Try to replace the frits/filters at each end of the bed.
The frits are the same material as the pre-filters, and will trap any small
particals causing back pressure problems. Clarify your sample by filtration
with a .2um filter or by ultracentrifugation.
I wash my columns with a few column volumes of 6M Guanidine HCl,
followed by a few volumes of 3M NaCl, a few volumes of water,
then 1% Triton X-100, followed by water, followed by a
0% to 70% ethanol to 0% gradient. Even with this wash protocol, I still
have to change the frits from time to time.
If you want to use SDS, try Li dodecy sulfate instead. It has a greater
solubility than SDS.
Ira Palmer
pe...@helix.nih.gov
NIAMS
Dr E. Buxbaum
> is there a good protocol for cleaning this column after i'm done with
This depends somewhat on what you have been loading onto it. If the
problem is precipitated protein, an urea gradient might help (and if it
does, it may be worthwhile to have urea in future samples too). With
lipid you may use a gradient 0-100% Methanol, followed by a
Methanol-Chloroform gradient and the same way back. If you have
particulates in your sample, you may want to think about appropriate
sample preparation in the first place (a few minutes in a table top
centrifuge can spare you a lot of grief). All buffers of course should
be vacuum filtered over a 0.22 um filter both to remove air and
particulates (one would not believe how much sot there is in analytical
grade salts).
With Pharmacia columns you should be able to remove the piston from the
top and change the net at the end of it, if that has become clogged. Be
carefull though not to introduce air bubbles in the process (ie have the
pump running while you remove the piston, so that the void fills with
buffer instead of air).
As I said, it depends on what the problem is.
Tobias Straub
wo...@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
>wo...@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...@mail.uni-wuerzburg.de
noone
>
> 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).
>
And do not only check the filter of your column but also the filter behind
the mixer (check if the high backpressure persists if you run the machine
without a column). In extreme situations , you may treat your column with
pepsin in a low pH-buffer (I know, proteases and columns are a scary
thought, but pepsin should be pretty inactive at neutral pH) to get rid of
clogged-up protein.
Run your column upside down to loosen the compressed material a bit while
you clean it
Hope this helps,
Peter