Have you tried reverse purification over the Ni-NTA column? That is the typical
next step in purification after His-tag cleavage. Or did you mean to say that
the impurities elute off with your cleaved protein during the reverse
purification? If this is the case, you could try adding a reducing a
Hi Theresa,
You could try lowering the pH down to 7.4 or 7. I have found that some proteins
bind very poorly at pH8 and higher. Hope this helps. Cheers!
---
Greg Costakes
PhD Candidate
Department of Structural Biolo
Hi Careina,
Your starting R/Rfree values seem just fine. The reason for the large disparity
after refinement is because the weighting factor is way too large during the
first rounds of refinement. For each round of refinement, have a few different
runs which test weighting factors between 0.01
When I was using BALBES, I had to download everything manually from the server,
one by one. However, you do have the option of running BALBES from within ccp4,
which will automatically save all of the files to a local folder .
If all you want are the files needed to continue refinement, then yo
My name is Greg Costakes and I am a graduate student for Cynthia Stauffacher at
Purdue University. I am attempting to solve the structure of an 18kDa protein
domain using molecular replacement to obtain proper phasing (44% identical /
78% homologous to an existing structure). Crystals diffracted
