Native PAGE Help - (Jul/31/2007 )
I am trying to run a native gel and the protein of interest (400kD, pI of 5.4) doesn't seem to migrate extremely well. I have tried
380mM Tris, pH 8.8
8% Acrylamide
0.1% APS
0.06% TEMED
for the resolving portion
and 380mM Tris, pH 6.8
5.1% Acrylamide
0.1% APS
0.1% TEMED
for the stacking portion.
On an Invitrogen Tris-Acetate pre-cast gel, the protein and marker seem to run well, but not on this setup described above. Does anyone know what is wrong with the setup described above?
Well, maybe I'm wrong but for me a native gel is the one that has one concentration of polymer and the same pH a long the gel and what you describe above is a discontinue gel. If you want a discontinue gel the concentration of the Tris is wrong. The concentration of the TRIS in the stacking should be lower than the resolving, also the concentration of the TEMED and APS shold be lower. Fro the resolving 100microL of APS and 25 microL TEMED per 20mL should be fine and 50microL of APS and 5 microL TEMED per 5mL for the stacking (thats enough for 2 mini gels). Hope this help.
You are much better off using pre-made 4-20% gradient gel if you can get one.
380mM Tris, pH 8.8
8% Acrylamide
0.1% APS
0.06% TEMED
for the resolving portion
and 380mM Tris, pH 6.8
5.1% Acrylamide
0.1% APS
0.1% TEMED
for the stacking portion.
On an Invitrogen Tris-Acetate pre-cast gel, the protein and marker seem to run well, but not on this setup described above. Does anyone know what is wrong with the setup described above?
your main problem is that your gel is too concentrated for a protein as large as yours. you can use a larger pore separating gel (5-6%, stack 3-4%) or a gradient like genehunter-1 suggested.