Hi all,
I have been trying to run a native gel. After running the gel I see only some blurry bands. How can I obtain sharp band?
The MW of the protein is 18kDa and the theoretical pI is 5.54. How should I choose the conditions for the native gel? Now I have used 4% stacing gel (pH 6.8, 0.125 M Tris-HCl) and 7.5% separating gel (pH 8.8, 0.375 M Tris-HCl). Running buffer is 0.25 M Tris-HCl, pH 8.0. Is there any molecular markers for native gels?
Thanks!

maybe your sample has too much salt in?

For a 18kD protien, i would choose something like a 10-20% gel.

scolix is correct, you need to use a higher percentage gel.

you can also change your electrode buffer to tris-tricine. this is a better buffer for small proteins and peptides.

Now I can obtain sharp bands. Problem is now that the protein I study do not enter to the 12% resolving gel but stucks between the 4% stacking gel and 12% resolving gel. Why is this? Should I use lower persentage gel; 10%?

are you still using the same buffer system as in the first post?

your running buffer looks wrong, the pH should be higher.

you can try a 5-15% gradient.

your protein may be aggregated.

I have now used Tris-glysine running buffer that has pH of 8.6.

I now used 10 % resolving gel and my protein still do not enter to the resolving gene. However as I used 7.5% resolving gel, after running 2 hours the gel was empthy. I quess my protein had gone rapidly throuhg resolving gel, is this possible? I am afraid that I can not used gradient gel because they dont sell precast gels to the machine that I am using so maybe I just have to optimise.

It can be that the protein is dimer in native form but I dont have information about that; This is recently found protein.

pour your own gradient. if you don't have a gradient mixer then borrow one (don't let the gel polymerize in the mixer, it's a pain to clean, reduce the temed and persulfate and wait longer for it to completely polymerize).

did you try 10% with the pH 8.6 buffer?

I tried 10% gel with pH 8.6 buffer. After 3 hours the protein is still between resolving gel and stacking gel. The protein can be homodimer so the real size of the protein can be either 18 kDa or 36 kDa.

for some reason your protein seems to be aggregating at the stack-run interface.

to determine if there is a problem with the stack, try running without the stack. make your load volume low so that you don't experience too much band spread during the run (a gradient gel would sharpen the bands some).