I am doing a Ni-NTA chromatography purification of my protein.
I noticed that air bubbles get caught within the column when I've poured my resin into it and I read somewhere that I can use 20% ethanol to get rid of the air bubbles. However, my resin is equilibrated with solubilization buffer before packing the column. Will the 20% ethanol de-equilibrate my resin? Please help.
I would not recommend this procedure in your case. While 20% ethanol washing do eliminate air bubbles sometimes (for example in FPLC pumps), its efficiency in the column chromatography is not that obvious. I would recommend to repack your column in your equilibration (solubilization) buffer. And yes, 20% ethanol de-equilibrate your resin.
You can pack the resin in the column first, and then equilibrate it with your solubilization buffer (try to make all procedures at the same temperature).
Hii
U r also equilibria yr column with running buffer also and regenerate yr with 50 mM Naoh also use.
Hi Barney,
Bubbles appear when air dissolved in your buffers comes out of solution. The reason can be a change in temperature (air is less soluble in water at higher temperatures): if you keep your resin at 4°C and run your column at room temperature, that might be the reason of your problems. To avoid this, leave the necessary amount of resin at room temperature overnight and stir it gently to remove air bubbles that have formed before packing the column. If you want to rescue your column that has been already packed, and if the bubbles are small, you can also degas your buffer under vacuum (while stirring with a magnetic barrel, this greatly speeds up the process) and flush your column with it. Otherwise, follow Viktor's advice. Another reason why you should not use 20% ethanol directly on your column is the risk that salts present in the buffer precipitate with ethanol. You should always include a wash with water between buffer and ethanol.
Olivier
hi!, you should always degas all your buffers under vacuum, while stirring with a magnetic barrel, before you use them on column purifications.
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