protein expression (temperature issue) - trying to express soluble protein at lower temperature (Oct/25/2009 )

simlez on Oct 31 2009, 07:31 AM said:

if u hv mass spec facility, cut the bands , trypsinize and identify on MALDI.
all the best.

DRN on Nov 1 2009, 12:13 PM said:

further, 'bout the inclusion body prob, u might hv to pull it out by urea denaturation method , or maybe try cloning the gene into some other vector like chaperon co-expressing one???

Hey,
Is it possible to upload a gel image of the result..?
Also mention the desired protein size and the other conditions.

Perhaps, then it would be easier to identify the problem.

regards,
Kaushik

DRN on Nov 1 2009, 12:17 PM said:

simlez on Oct 31 2009, 08:31 AM said:

Do an ELISA (if your lab has ) or a HPLC (for this u need to have the standard to match the Retension times)!!!

DRN on Nov 1 2009, 05:47 PM said:

... or clone as a different fusion protein. Try a His tag or GFP fusion

cn u say what's ur protein size.

Use a tag!
If you are having that kind of problems it's the best way: it's easy to clone, easy to identify... and easy to remove...
HPLC, mass spectrometry, western, ELISA....c'mon... each time? Add any tag (6xhis?) and if you need it, you can remove it later... but maybe you don't need it!

Not sure whether is it relevant but you can try cell free expression...