I purified a C-terminally His-tagged nanobody using Ni-NTA. After labeling the nanobody with a alexa dye, when I was trying to get rid of the free dye using Ni-NTA, I found ~30% of labeled nanobody is trapped in Ni-NTA (Elution buffer contains 20 mM Hepes, 150 mM NaCl and 250 mM imidazole). I am not able to recover that fraction even after using 500 mM imidazole in the elution buffer. I don't want to apply dropping the pH in the elution buffer as this might not be good for the stability of the nanobody. How can I get rid of this? If anyone experienced this problem, please suggest me.
Increase NaCl conc and see if it improves. Do you see some kind of precipitation?
Hello Rajdeep
You may use 300 mM NaCl in your buffers (Equilibration, Washing and Elution). Alternatively you may low the pH in your elution buffer and immediately restore the pH by keeping NaOH in tubes (where you are collecting your fractions) followed by dialysis against desired buffer.
Hope this will help.
I would also recommend increasing your salt concentration to try and remove the nanobody completely from the Ni-NTA resin.
However, if you're only using the Ni-NTA as a way to remove excess dye, I would suggest just using a small spin desalting column. This may work for you depending on the volume you are using. We have used
Hey Omar, I didn't see any precipitation. Thanks to all for your suggestions.
I would suggest you to use a high salt concentration like 500mM of NaCl in your washing buffer and then use a suppose 300mM of NaCl for a second wash and then finally elute out in 150mM NaCl as too much NaCl in your elution buffer is not a good idea.
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