I am using a HisTrap HP 5ml column from GE for protein purification and I accidentally pumped air into it. I was wondering if there is way to remove air from this column?
Degas well your running buffer, water or 20% ethanol and pump it through the column from below. Yes, from the down exit while keeping column in a vertical position. Keep doing it until all the air is out and even the small air bubbles have dissolved. And be careful next time!
1 - I would ask GE
2 - why do you need to remove air, if oxygen does not damage your subject then no sense to remove air
Degas well your running buffer, water or 20% ethanol and pump it through the column from below. Yes, from the down exit while keeping column in a vertical position. Keep doing it until all the air is out and even the small air bubbles have dissolved. And be careful next time!
Thanks for your suggestions, So i pumped this Nickel column with 20% ethanol keeping the column upside down, but it seems that the column does not respond to the pressure like it used to do before. I usually run my his tag protein samples at 3ML/MIn on this column (since it is a 5ML column and the max. run possible was 5ML/Min) but now when i connected the peristaltic pump and I am using 20% ethanol to flush out air at 3ML/MIn, it doesn't seem its pumping out ethanol at the same speed, its very slow. So I am wondering if i can still use this column for purification? What do you guys think? I left it for an ethanol wash overnight.
if oxygen does not damage your protein then it is ok and you can run the column, you will just get not so sharp peak of your target protein
In my experience columns can be degassed by counter-flowing properly degassed water (0.22 um filtered and autoclaved/thoroughly vacuum pumped). Very low flow helps removing air bubbles.
Alternatively air can be "pumped out" by connecting one syringe at either end of the column using one to inject degassed water from one side whilst using the other to suck the buffer out. Repeating this procedure (you might need to pass 100-200 ml water through) can also recover columns totally filled with air, not just an air bubble. In this case pumping buffer out with the second syringe applies a strong enough vacuum to pull out the air as in a normal buffer degassing procedure. Supplying buffer with the first syringe avoids overpacking of the resin and fills the gaps left by the removed air.
HTH,
Marco
Hi Amit, running buffers containing detergents through the column might help dislodge stubborn air bubbles.
Hello Guys, thank you all for the valuable comments and suggestions. I finally realized that 20% Ethanol works perfectly well, although it didn't seem so initially and I had to leave the column running for a couple of days till all the air bubbles were out. Thank you all for your suggestions....Cheers.
By using syringe, pump 5 ml 20% ethanol through the column from below and it will remove the air bubbles.
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