>From what you describe cation exchange appears to be a viable option. With
pI' s of 6 and 9, you will want to pay close attention to the pH of the
column eluant. I'll refrain from recommending a specific vendor (I bet
Amos would be glad to jump in here) but suggest that you not use
polystyrene-based ion exchangers for this application. In my experience,
polystyrene anion exchangers interact with analytes via the hydrophobic
effect, i.e., they are mixed mode resins.
BTW- you may want to repost the message. Without including carriage
returns, I had to copy your file to my word processing program before I
could read it. Hope this helps.
Russ Lehrman
On 1/15, Brett Phinney wrote:
I have a pretty basic chromatography question that I hope someone can help
me with. I am trying to separate 2 integral membrane proteins. The problem
is that these two proteins have almost the same MW (approx. 56 KD each) so
SDS-PAGE does not work very well. I have an HPLC and have been trying to
separate these proteins using a RP-C18 column, but this also has not worked
so well, I think that the proteins may be too hydrophobic and thus are
sticking very tightly to the column and not coming off. I wanted to try
either ion-exchange or maybe a less hydrophobic column like a C4 or CN. The
proteins have pI's of 6 and 9 so I was thinking Ion exchange with a
non-ionic detergent such as octly glucoside may be the way to go.
What should I try? If ion-exchange is the most appropriate, what would be a
good column to try. Considering the cost I don't really feel like spending
$700-1000 dollars on a column that will not separate my proteins, although
I may not have a choice...
Thanks a lot in advance for the help
Brett Phinney
North Carolina State University
Phone (919) 515-5786
Fax (919) 515-2047
phinney@bchserver.bch.ncsu.edu