Hello everybody,
it could sound a little bit strange. My problem is that I have a large volume of a protein sample. But my capacity of the gel pockets are restricted. Now my question can I pour a gel with a long stacking gel that my volume will fit properly? Because I don`t want to precipitate my samples. Or will I get problems? I am not sure. Once I read one should have a long separating gel. My mw of my protein is about 180kDa.
Please help me.
Thanks
Tiffy

The short answer is Yes,
The wells could be long, if you know that your protein is 180kDa just use a gel concentration that wouls seprate higher mollecular proteins.
Just remember not to load to much protein.
Guy

PS.
If you intend to blot it for western do not cut the seperation portion of the gel.

Thanks guy,

is 50µg protein okay? Or is it too much?
Best regards Tiffy

PS It`s not really clear to me why I have to blot the stacking gel as well. Could you explain it please.

Happy to help you,
50ug is perfect, you can go up to 100ug with no problem.
You should blot with the seperating gel so if some of the protein had agregated it would be seen on the blot.
Guy