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.
with my loose agarose I usually centrifuge at low RPM and let the beads settle. Tip off the supernatant and then rinse a few times with water and 0.5M NaOH - should bring any residual mess off the beads for you (centrifuge and remove supernatant between each regenerative wash)
Do you get a pellet of particulate material above and/or below the layer of resin after the centrifugation? Since you are going to regenerate the resin anyway, dilute out the lysate to lower its density, centrifuge, pour off supernatant, dissolve particulates with alkaline 6M urea or guanidine HCl with EDTA, re-centrifuge and pour off the supernatant. You might want to repeat this a few times and then with water to prepare for the regeneration step. The guanidine may make the resin become partially transluscent but it will return to opacity with the water rinses. Depending upon the amount of resin, this should be fairly quick processing.
You might want some cleaning validation so check the supernatants for endotoxin, A280/A260 (protein and nucleic acids). If you're using Qiagen Ni-NTA Superflow, the resin is 6% cross-linked at 100 micron beads and relatively more rigid allowing for rapid flow near 100 psi. So you can rapidly do your process once you get the particulates solubilized. BE SURE TO CHECK THAT ALL UNBOUND NICKEL IS COMPLETELY REMOVED FROM THE RESIN AT THE END OF YOUR REGENERATION. There are a number of specific nickel assays available including a simple precipitation test.
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