ugly bands on sds-page of high mw protein (mTOR) - (Nov/28/2006 )
Hey everyone. Any help on this would be great. I am trying to look at mTOR, which is 286 kD. My bands for other proteins such as Akt or GSK (~60kD) look nice and sharp and thin. But when it comes to mTOR, my band is broad and smeared and quite ugly. Why is this, and what can I do to get sharp bands? I am loading 30 or 60 ug protein per lane. Separating gel at 5%, stacking at 4%. 60V until reach separating, then increase to 100V for about 1 h or so (until dye runs out) on a mini-gel. Don't know what I am doing wrong. 
Here are some thoughts on this, see if it works: 1) It has a lot to do with the quality of the gel that you made, esp on the top portion of the gel. O2 exposure is not good for polymerization. Do use degassed gel solution and cover it with butanol (saturated with H2O) during gel casting. 2) try to reduce the voltage applied and run it for longer period of time. 3) any chance that this TOR is glycosylated? Increased sugar content could make band broad or more heterogeneous.
the concentration of detergent is important too.
when I use triton X-100 1% (load around 20 µL, the migration is bad in the upper part)
with only 0.1% the migration looks fine.
how much detergent do you have?
I doubt that you have got nice and sharp bands of Akt/PKB or GSK in a 5% AA separating gel, actually they will co-migrate with the dye front
I do not degas, but I do overlay with ethanol. I will try degassing next time. How is this done, btw? I have never done so before. I do not know if mTOR is glycosylated--will look into. Thanks!
when I use triton X-100 1% (load around 20 µL, the migration is bad in the upper part)
with only 0.1% the migration looks fine.
how much detergent do you have?
Detergent in my lysis buffer? I use 1% Na deoxycholate, 1% NP-40, and 0.3% SDS (modified RIPA). Should I be using a different lysis buffer?
Hey everyone. Any help on this would be great. I am trying to look at mTOR, which is 286 kD. My bands for other proteins such as Akt or GSK (~60kD) look nice and sharp and thin. But when it comes to mTOR, my band is broad and smeared and quite ugly. Why is this, and what can I do to get sharp bands? I am loading 30 or 60 ug protein per lane. Separating gel at 5%, stacking at 4%. 60V until reach separating, then increase to 100V for about 1 h or so (until dye runs out) on a mini-gel. Don't know what I am doing wrong.
I doubt that you have got nice and sharp bands of Akt/PKB or GSK in a 5% AA separating gel, actually they will co-migrate with the dye front
Actually, I am not looking at all of these proteins on the same gel. I use a 10% gel for Akt and GSK. Just thought I'd mention that I am successful at looking at other proteins.
the concentration of detergent is important too.
when I use triton X-100 1% (load around 20 µL, the migration is bad in the upper part)
with only 0.1% the migration looks fine.
how much detergent do you have?
Detergent in my lysis buffer? I use 1% Na deoxycholate, 1% NP-40, and 0.3% SDS (modified RIPA). Should I be using a different lysis buffer?
I don't think so. Maybe you could try to make some more concentrated extracts, so you could load less detergent on the gel the next time?
what's the amount of protein you are loading?
the concentration of detergent is important too.
when I use triton X-100 1% (load around 20 µL, the migration is bad in the upper part)
with only 0.1% the migration looks fine.
how much detergent do you have?
Detergent in my lysis buffer? I use 1% Na deoxycholate, 1% NP-40, and 0.3% SDS (modified RIPA). Should I be using a different lysis buffer?
I don't think so. Maybe you could try to make some more concentrated extracts, so you could load less detergent on the gel the next time?
what's the amount of protein you are loading?
I loaded 60 ug for one cell line, 30 for the other. I need to make more concentrated extracts anyway, so I will try this. Thanks.
you may be able to sharpen the bands if you run a gradient gel. even a shallow gradient (eg-5-7.5%) should improve your bands.