During protein purification, I have some hemin precipitate on Superdex 75 column, which made column greenish on the top. Does anyone have experience to clean it?
If it is just on the top, you could remove the visible precipitation layer (provided you have a column you can open and re-seal safely).
In general running a NaOH-solution for a long time might (! - as we do not know the cause of the precipitation) dissolve the hemin again. Adding triethanolamine will likely help. (Be sure that the waste goes really into a waste bucket and not into some beaker you want to re-use for really clean work.)
I cannot give you the 'best' concentration, but this would be my first attempt: 20 mM NaOH, 100 mM triethanolamine. I would prefer triethanolamine and not the hydrochloride, but that probably will not make much of a difference.
HTH
Christian
Either remove the top layer of beads
or do repeated injections of up to 1M NaOH in reverse flow (e.g. For a 26/60: 5ml, NaOH, then 10ml buffer, then 5ml NaOH , .. ).. GuaHCL injections might also work. The reverse flow prevents that the stuff bleeds through your column and eventually clogs the bottom filter, which is way harder to fix....
I did try some wash suggested. So far, no one really wash off the stain completely. I tried reverse flow at room temperature with 1M NaOH, 6M guanidine (turned the color form greenish to reddish), 10% ammonium hydroxy, ethanol, and isopropanol.
Can I use 100% ethanol? The maximum concentration allowed to superdex 75 is 24%, right?
The best way is open the column & remove the percipitate.
1M NaOH , 30 % isopropanol in downward and upward direction 2 times the column value followed by intermittent water wash will help
I have had that problem in the past with my S200 and running 0.5M NaOH inverted was enough to clean it. I didn't have a stain though, I just knew I had some hemin stuck to it because my proteins were running different.
Wash the column with 1M NaOH and water. Now run 25% EtOH followed by water, repeat EtOH-water for 3-4 times. It works. We had same problem with superdex 200, This method helped to remove lots of heme. At the end, just open the top piston and remove little bit resin, place a new filter. Wash it again with water. It will be totally clean.
Just now
You could use 1M imidazole to get rid of heme (which is quite hydrophobic otherwise) and then wash the column with loads of water.
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