What type of native gel (pH/acrylamide %) do I use for a protein with the pI=9.0 - (Jan/10/2007 )
I need to run a native gel to look for dimerization (trimer,tetramer, etc) of a protein with the Molecular weight of 113kD and a pI=9.0. I've heard and read different things about what I should do. Should I run an acidic native gel system (running from + to -). What pH should I be trying to reach? What percent gel should I use? Should I use a stacking gel or should i be continuous? If I include a stacking gel, what pH should that be? If I use an acid native PAGE system, should the sample buffer be acid too? If anyone could help me answer these questions that would be great or reference me to a protocol that would be good also. Thank you for your time. Kevin
yes, you should use an acid gel system (you could also use a neutral pH system, but acid would be better).
see this thread for an acid gel buffer system:
acid gel
for your size protein a 7.5 - 10% (not referring to gradient) gel will probably be okay.
just add glycerol and tracking dye to your sample and it should run okay.
Would bromo phenol blue work as a tracking dye in a acid gel, I am concerned that it will run out the otehr way when I reverse the electrodes (+ to -)... Thanks, Kevin
it's been a very long time since i've used acid gels so i don't remember for sure but i think we used bromphenol blue (it may turn yellow) but you could try to find a cationic dye that may work, instead.
DO you have access to an FPLC system? If so, you could try running your protein down a gel filtration column that has been calibrated with a range of different proteins. This will also give you ideas of the ratios of different forms, and if you change the salt concentrations, or salt type, how strong the interaction(s) is/are.
If you really want to find out about the state of the protein, you can use analytical ultracentrifugation. It takes about a week to run the expts, but you can pull all sorts of good data out.