Hello, I am purifying a his tagged protein on a hi trap column charged with nickel. I noticed that after I added the buffer over the column it changed from green to brown. The buffer does have 1mM DTT in it. Will this affect the proteins ability to bind to the column?
Thank you
0
2 replies to this topic
#2
Benita
-
- Active Members
-
- 18 posts
member
0
Neutral
Neutral
Posted 10 June 2010 - 12:48 AM
mcb56, on Jun 9 2010, 11:20 PM, said:
Hello, I am purifying a his tagged protein on a hi trap column charged with nickel. I noticed that after I added the buffer over the column it changed from green to brown. The buffer does have 1mM DTT in it. Will this affect the proteins ability to bind to the column?
Thank you
Thank you
Yes you washed the nickel from your column. You have to recharge it with nickel solution again. Theres also a part about that in the manual.
Ciao!
#3
parado
-
- Active Members
-
- 8 posts
member
0
Neutral
Neutral
Posted 13 June 2010 - 01:05 AM
Benita, on Jun 10 2010, 04:48 PM, said:
mcb56, on Jun 9 2010, 11:20 PM, said:
Hello, I am purifying a his tagged protein on a hi trap column charged with nickel. I noticed that after I added the buffer over the column it changed from green to brown. The buffer does have 1mM DTT in it. Will this affect the proteins ability to bind to the column?
Thank you
Thank you
Yes you washed the nickel from your column. You have to recharge it with nickel solution again. Theres also a part about that in the manual.
Ciao!
i think it is because the Ni ion is reduced not eluted .. because of the DTT ..
if you want to elute the Ni it should be EDTA which is needed when you want to regenerate you collomn
