FPLC Column Cleaning Protocol?
Wed Jul 22 07:46:56 EST 1998
In <6p3png$ec0$1 at news.fas.harvard.edu>, wong5 at login2.fas.harvard.edu (Donny Wong) writes:
>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.
>wong5 at fas.harvard.edu
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.
pelip at helix.nih.gov
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