Dear all,
I have two questions:
First, I was trying to crystallize a complex of two proteins. Both proteins has
been crystallized before. The two proteins bind to each other based on Biacore
study, but they didn't form a single peak on gel filtration. When I mixed them
at 1:1 ratio, the crystals I got contain only one of the two proteins. I was
suggested to increase the ratio, for example 1.5:1, to increase the probability
of co-crystallization which I will try. But I do want to hear if there are
other possible ways to try. What would you try if you were in my situation?
Second is about reusing of Ni-NTA resin. According to Qiagen's instruction,
after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first
with 0.5M NaOH, then with your own buffer. After that the resin is ready to be
reused until it needs being recharged. But my question is: Once immidazole
competes with His-tagged protein and binds to Ni-resin, how can immidazole be
rinsed off with the same buffer(usually pH is above 7) one uses to purify the
protein?
Thank you for any suggestion or comment.
Min
