Since you're seeing the MBP band, it sounds as if you're getting some cleavage. If there were no cleave you would only see the fusion and TEV bands on the gel.
It may be that your protein is not stable/soluble without the MBP fusion. Different buffer conditions may help if your protein is being lost post-cleavage. The efficiency of cleavage may also be aided by different buffer conditions. Since you didn't report the buffer you're using, it's impossible to judge if it is appropriate for maximum TEV activity. Cynthia Sent from my iPhone On Nov 4, 2012, at 10:24 AM, "rana ibd" <rna19792...@yahoo.com<mailto:rna19792...@yahoo.com>> wrote: Dear CCP4 I am having a problem with cleaving my fusion protein and I would be grateful if you advice me regarding this situation, I have an MBP-DHBx fusion protein and I am trying to cleave it using TEV protease, I have tried different ratios and different temperatures with different incubation time but still it will not cleave, all I observe on the gel is the bands of the fusion protein which is 59kDa and the MBP which is 42kDa and the TEV protease which is 27kDa and no sign of the DHBx which is 17kDa,I have also checked the sequence if there was any problem but I could not find anything unusual the sequence was fine , so if you have any suggestions regarding this situation I will be thankful Best Regards Rana
