Hi...
I seem to be in a very big trouble. My His columns look cracked after my last purification. In another column, the materials seem to be pushed. How is it possible and is there anything I can do to fix it? I would also appreciate any advice for the future purifications.
This is a His HP column. I always load lysate containing 8 M urea as my protein is in the inclusion bodies. The loading is at 0.5 mL/min (the slowest the ÄKTA Start can go). I think there might be some pressure issue or clogging as I always face trouble with the lysate everytime I do a purification.
The cells were lysed using High Pressure Homogenizer for 4 cycles, centrifuged at 14.500 rpm, and the supernatant filtered using a 0.22 um paper filter (as it's not possible using a syringe or vacuum filter). Unfortunately the cells can only be solubilized with 6-8 M urea, and guanidine is not an option as it is expensive to buy (we have limited budget).
Thank you so much.
Best,
Lieke
Having a column bed separate like that typically means you have had a backward flow. Opening your inlet tubing while the system was still under pressure can do that (for example, doing manual sample loading on top of the column before the system pressure dropped to zero, leak in the injection valve, or the tubing popped out during the run, etc.), as is stopping the flow before high concentrations of buffer or urea has been flushed out of the system. You should always ensure the flow has stopped and any residual pressure is gone before opening up the system, and all buffer salts and urea get flushed out before stopping the flow (can take quite a while at 0.5mL/min).
You can take a cork ring or a big rubber stopper to gently knock on the side of the column to get the stationary phase down, then gradually increase the flow rate to reach ~80% of the maximum pressure rating of your column while continue knocking, and gradually reduce the flow back to zero. You can then do a test run with a known sample. If the performance is acceptable, you are all good; If not, it would be time to confess to the prof and buy a new column :(
Hi Lieke,
I agree with Gang Chen with regards to the reason for this situation. You can use the method of Gang Chen to get rid of the crack, which should work.
In addition to that, a convenient and easy method for regeneration of a dry column or a column with air bubbles, which works fine in our labs, is putting the column without stop plugs in a beaker, and filling the beaker with storage buffer (20% ethanol), so that both ends of the column are in the buffer. Then put the beaker in an exsiccator and apply vacuum, e.g with a water-jet vacuum pump. The air from the column will be removed directly.
This can also help in your case to remove inhomogenous parts of the column.
And a bit of advertising on our own behalf: Cube offers XL resins for viscous media, which generate a lower back pressure as they consist of larger particles and therefore also have larger interspaces. They are available as ready-made columns with Ni-NTA, glutathione and Ni-INDIGO, an improved NTA resin, among others.
Best, Roland
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