Recent replies to "Unable to express protein in full-length form"

Reply from anonymous

anonymous — Tue, 26 Aug 2008 17:29:26 -0000

Did u check the DNA seq to see any stop codon mutation in the middle of the gene?
And use a brand new protease inhibitors when you purifying the protein.
What method did you use to detect your protein (CB stain or Western Blot?) I would use western blot because sometime the protein expression level might too low that CB stain cannot detect.

good luck

Reply from Karl Erlandson

Karl Erlandson — Wed, 23 Jul 2008 19:30:19 -0000

As they said before, add protease inhibitors—PMSF/AEBSF or Complete tablets, benzamidine or even a mixture of them. Add them when homogenizing your cells, add again after breaking your cells, and add them again if you’re dialyzing at the end. You should also try doing your purification on ice or in a cold room.

If you have a tag on the C- or N- terminus, you can do a western to determine which end is degraded.

To test if it’s degrading itself… knock out the active site residue(s) of your protein and see if you still have this problem.

Reply from anonymous

anonymous — Thu, 29 May 2008 23:03:49 -0000

Have you also tried including protease inhibitors in your solutions? If you do not express many proteins you should probably read this paper from structural genomics: PMID: 18235434

Reply from Swetha Suresh

Swetha Suresh — Thu, 29 May 2008 12:11:31 -0000

hi
have you checked for activity? because once i was purifying my protein, the stop codon wasnt strong enough so I had many lengths of proteins and did think that my protein was being cleaved.

Reply from Asrar Alam

Asrar Alam — Fri, 16 May 2008 10:38:21 -0000

Thanks,
I haven’t tried expression in mammalian system but I am trying it in baculovirus expression system.

Reply from Raphael Roduit

Raphael Roduit — Fri, 16 May 2008 05:32:06 -0000

Hello
Did you try to express this protein in mamalian cells…293T cells or jurkat?
Regards
Raphael Roduit