I would like to run three different purified proteins on the same
native gel. They are all have a predicted isoelectric point below
7.0, thus a native gel run in basic conditions should suffice;
however, I am having trouble getting one protein with an isoelectric
point of 6.7 to enter the gel.
I am using an pH8.8 continuous gel (no stacking gel). I abandoned
using the pH 6.8 stacking gel because I thought it was too close to
the IP of my IP6.7 protein, and was the problem. I"ve also tried
adding BME to my IP6.7 protein to try and abolish some of the
tertiary structure which may influence the IP of the protein.
Is it possible to run a native gel under basic conditions, but jack
up the pH of the running gel and buffer system, to say pH9.5 to try
and make my predicted IP6.7 protein more negatively charged?
One complication is that I would ultimately like to use this system
to show that two of these proteins bind each other by running them as
a mixture on a native gel. So, raising the pH too much may interfere
with this assay. Are there any other ways to make a protein more
negative without interfering with its capacity to interact with