The protein extracts are frozen at -80 degrees, and we have tried to quantify by the Lowry method both before freezing and just after thawing and before loading the gel. Even if we quantify the total protein and load the same amount of protein on the gel, the loading control band always appears uneven, we have tested b-actin and vinculin.
1. Could the treatment actually affect expression of these proteins?
2. Maybe try a different quantification method? Maybe there is some error with the quantification?
3. Error introduced by pipetting during sample prep or loading the gel?
4. Problem with transfer? Sometimes the transfer will occur unevenly.
If you do a ponceau stain on the membrane right after transfer you might get an idea of what is happening. That should indicate if the amount of protein being loaded is actually even, and/or if there is an uneven transfer issue.
- Badges
- Science method