Elution problens - My protein is not eluting in urea 8M buffer ... oh shitt!! (Jun/10/2009 )

Hi, I'm a brazilian Ms.c student. My fusion protein used to elute on urea 8m buffer, but, recently it isn't!!
I made a westernblotting and it is still fixed on the resin Ni. I tried 3 times to elute it , but no lucky!!!
So, I need your help!


Another question: i'm going to use this fusion protein on ELISA experiments, which buffer is the best to store the protein. Could the protein under denatured condition be better to use on ELISA, because of the antibody access??

thanks a lot !

are you using fresh or old buffer?

if fresh, did you add everything to it that belongs?

did you heat it to dissolve the urea? if so, you may have decomposed the urea.

if old, how did you store it? the urea may have decomposed.

you may be able to elute your protein with edta (in a suitable buffer). this will strip the nickel from the resin so you will have to recharge it with nickel before reuse. the nickel will come off with the protein.