NI-NTA colunm purification - no bands after purification (Jul/18/2006 )
I expressed my protein (bacterial toxin 11kDa with His Tag) in pLysS at 30 C. I had low expression but I could see the band in SDS PAGE and also in Western (with anti HisTag). I tried the purificacion in Ni -NTA superflow Quiagen column but I found no bands in SDS PAGE after purification. What can I do? I need a urgent help, please!!
-dcc-
It sounds as if the protein has added affinity for the Ni-NTA resin independant of the His tag- the first thing to try would be to increase the concentration of imidazole in your elution buffer.
What imidazole concentration were you using to elute with when the protein did not appear in the eluate?
-holiday-
QUOTE (dcc @ Jul 18 2006, 01:04 PM)
I expressed my protein (bacterial toxin 11kDa with His Tag) in pLysS at 30 C. I had low expression but I could see the band in SDS PAGE and also in Western (with anti HisTag). I tried the purificacion in Ni -NTA superflow Quiagen column but I found no bands in SDS PAGE after purification. What can I do? I need a urgent help, please!!
Could you give us the photo after purification (conataining total protein; flow through; wash;elute)?
Firstly, you must find fraction containing the target protein?
-Brainzhang-