Hi, I run this gel using 3 different layers:
-the upper layer: 4%T 3%C stacking gel
-the middle layer: 10%T 3%C spacer gel (1cm)
-the lower layer: 16%T 6%C separating gel
-voltage: 60V until the sample enter the lower layer, then adjusted to 100V until finish
-reducing sample buffer was used, heated 80 degree C for 5 mins
-sample volume loaded (10,8 and 2 microlitre)
-all the electrode buffers were pre-cooled.
However, when i destained the gel i could not see the lower molecular weight proteins and i have realize that when i was running the electrophoresis, the tracking dye (commassie blue G) become a faint thick band after entering the lower layer of separating gel, this make me hard to define where the dye front moved.
Attached are the gel image.
This protocol recommends to use urea for hydrophobic proteins
https://info.gbiosciences.com/blog/tris-tricine-sds-page-what-is-it-and-how-to-perfrom-it
- Badges
- Science topic
- Similar topics
- Gels