I have recently started doing SELEX, and this is an outline for it:
Wash beads (I'm using Ni-NTA for a His-tagged protein).
Incubate with protein for 30 min.
Wash off unbound protein.
Add RNA.
Collect flowthrough.
Wash off unbound RNA
Elute RNA with Imidazole (400mM)
Strip beads.
I'm measuring the protein with each fraction using a Bradford's assay, and I found that I seem to be abut 40% short of the amount of protein I originally put in. Could anyone point me in the direction of where it might have gone wrong?
Also does anyone have a standardized SELEX protocol they could share?
:)
12/5/17
Dear Surabhya,
I've never done SELEX; however, I have done His-tagged protein purification using Ni-NTA. Is it possible that the concentration of imidazole you are using is too low to elute the protein? Perhaps you can try a higher imidazole concentration.
I hope this information/suggestion helps you.
Bill Colonna Center for Crops Utilization Research, Iowa State University, Ames, Iowa USA [email protected]
I did that too by adding a 1M imidazole elution step. Apart from this I also stripped the beads with EDTA
Hiw did you construct the calibration curves for the original samples and eluted material?
Surabhya,
As mentioned above the efficiency of elution from Ni-NTA beads varies among proteins: to this end did you test it without the addition of the RNA?
If not I would suggest to test that in order to find the best concentration of imidazole that presents the highest recovery, and repeat the the experiment.
Surabhya Balu I'm trying to do protein SELEX right now with his-tagged beads and am having trouble with DNA yields. Did you ever figure out a good protocol that works for you? I've tried imidazole vs. heat elution, but I've never been able to recover all of the DNA that I put into it (I think some isn't eluting).
Thanks!
- Badges
- Science method