Sorry for the off-topic, but I need some help. I have a protein with a HisTag. I need to deglycosylate it prior to crystallisation, as the glycosylation trees interfere in the process. As I didn't want to change the buffer the protein is in, I use the enzyme EndoHf (which has an MBP-tag), and after incubation I load the whole sample onto an MBP-trap to obtain my protein in my buffer, and then I can elute the EndoHf separately with maltose.
It worked perfectly the first time (freshly prepared buffers, new columns, fresh enzyme). The second time I did it, I didn't get the EndoHf peak in my chromatogram, and a gel confirmed that it was still with my protein. I checked everything and realised that the buffer pH had dropped slightly below 7.0 (the column specifications say it should be above that value), so I changed the buffer pH, and finally it worked. After that, I have always checked the pH of the buffers prior to incubation, and I leave it around 7.5. However, in the following assays I've had the same problem (both proteins coming together), and I know it's not a problem of pH any more. I have asked the company of the enzyme and they say it is quite strong and there shouldn't be any problem with the tag, even if it has been melted/frozen several times. I also followed the instructions of the column, I have regenerated it, and I have used a fresh one, with the same results. Now I don't trust this method (I lose time and protein) and do it with a HisTrap, even if that means buffer-exchange afterwards. But I'd like to know what could be happening. Any ideas? -- Saioa Urresti, PhD. Postdoctoral Research Associate York Structural Biology Laboratory Department of Chemistry University of York Heslington, YO10 5DD, York, UK