I am attempting to measure nuclear and cytosolic proteins from human muscle. Currently, I am having trouble with my nuclear western blots. As you can see from the attached image The first and third 5 lanes of the stain free gel (Bio Rad Crit TGX 4-20%) are the nuclear proteins and the second and fourth 5 lanes are my cytosolic proteins.
I have attached another image, from this Western I was able to validate the the purity of my blots via H3 and LDH. However, you can the H3 protein streaks down the membrane, this has lead to an inability to quantify my Nuclear westerns.
I loaded 12 uG of protein in each lane.
Ran @ 80V for 2.5 hours.
What could be the issue? Do I need a different running buffer for the nuclear proteins? Running buffer used: Tirs-Glycine-SDS
Any suggestions?
- Badges
- Science method