Dear all,
I accidentally mixed Ni-NTA beads with E. coli cell lysate but has a problem with separating them. Is there any way to separate them so that I can regenerate Ni-NTA beads for future use?
I tried centrifugation at low speed (800 x g, 5 min) but didn't work.
Thanks in advance.
Hi there,
The low-speed centrifugation should be OK. How viscous is the cell lysate? Otherwise you may load the mixture onto an empty purification column like the ones from Biorad and settle, elute and wash with gravity.
If some protein on your lysate has a His tag you must to elute with a buffer containing imidazol, if there is not the case the best is to do as Dominique suggests, load into a column and elute by gravity the lysate.
You could also use 25µm filter plates (BS4 74-5558 - http://www.sdr.com.au/filterplates.php) and centrifugation to separate the beads.
You've essentially and accidentally set up a batch purification. You should be able to recover the beads by centrifugation at about 500RCF, or as suggested by loading them into an empty column. I'd recommend you load the beads onto a small column for elution step.
@dominique the lysate wasn't viscous at all, and yes, thx for your suggestion I will absolutely try that :)
P.S. I am not eluting the protein out of the column. But instead, there's a clump on my Ni-NTA beads. Some are very sticky and forms a clump with the beads when I mix them, and some becomes soluble after mixing.
- Badges
- Science method