Hi all,
We have HiLoad Superdex 200 16/60 Gel Filtration column in our lab, which have some 'Sticky Proteins' and protein aggregates bound to the column. As such, I wish to perform some rigorous cleaning of the column to get rid get rid of these unwanted debris on the column?
I have done some reading from the column manufacturer manual (GE) and they have recommended 30% isopropanol and 70% of ethanol together with high denaturing agents (Ie. 6M Guanidine Hydrochloride/8m Urea).
Based on your experience and opinion, what would be the best approach/cleaning solutions.
Thanks.
Most of the time, 30% isopropanol and 70% of ethanol together with high denaturing agents (Ie. 6M Guanidine Hydrochloride/8m Urea) will work better.
You can also reverse the column and run with 0.2 ml - 0.5 ml. And an alternative is 0.5 - 1M NaOH
Hi there,
The cleaning in place procedure is described here:
http://www.smansy.org/lib/exe/fetch.php?media=public:hiload_16_60_200_pg.pdf
with more options there:
https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314716762536/litdoc71705900_20110830182107.pdf
You can also consider NaOH treatment with reversed flow in order to eliminate insoluble at the top of gel bed. Avoid mixing chaotropic agents and alcohol as I'm not sure about high solubility of the former in the latter...
Hi Saranpal!!!
in our lab we frequently use the following to clean the gel filtration column. First you need to run double column vol of the filtered Milli-Q then run Milli-Q with pH=2 or 3 then inject pepsin 1mg/ml according to sample inject procedure. Run the column at 0.1 ml/min flow rate. next day you should run the column with 0.1 mM of acetic acid then with Milli-Q. You should wash your column with 0.1 mM acetic acid after 3-5 run. use these steps, hope these could help you.
gud luck.
1 M NaOH with very slow flow, but check on the stability of the column material against alkaline conditions.
This will crack aggregates and the like.
The fragments can then be washed out with 30 % isopropanol.
You can first wash with Guanidine Hydrochloride, then with 0.5 M NaOH, then with 70% ethanol and 30% isopropanol for storage of the column. When you use it, then first wash with MilliQ water and then wash with TRIS buffer with high concentration of NaCl and then equilibrate with your working buffer. I hope this helps.
Regards
If you are sure about the sticky proteins, then i shall go to denature them and chop them out first before trying to wash them out. There are many nice (but brutal) tricks provided above. Just be careful. Some of them are meant to be the last resort.
Hi. Does it work at the end? Can you tell me how to use pepsin cleaning? Because its said to inject pepsin solution and incubate it overnight in RT or 1 hour in 37C. How to inject it? Where to inject? Thanks before
Hi,
first with 30% isopropanol 1 CV, wash with 1 CV H2O
- Wash with 0.5 M NaOH 1CV, wash with 2 CV H2O
- additional cleaning using 500 mM acetic acid can be useful. Wash with 2 CV H2O
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