During my western blot my protein gives band nearly between 42- 52 KDa.
I want to use beta actin or beta tubulin as my loading control. But, unfortunately it also gives band in the same region. How should I perform my western?
Please Help me.
GAPDH has a molecular weight of 37kDa. Maybe you can use that instead?
Dr Pooja
you can adopt 2 different approaches
1. Use single primary antibody at a time to detect w. blot and then strip the same blot, followed by second primary antibody.
2. Use both primary antibodies at a time but from different sources, say one from mouse and second from rabbit. Then detect blot on LI-COR
Best of Luck
If you get the normal and classical way, you can load GAPDH and check if it not changed with the time. Other option is to stain the membrane with Reactive Brown or another chemical and use the band close to this molecular weight but more stable. Since many intervention produces an increase in many proteins I recommend you this last one.
The simplest answer is to use a different loading control antibody. GAPDH or COX IV are both sufficiently separated. Alternatively, multicolor detection using fluorescence or IR (e.g., LI-COR imaging as Ajaz said) is an option.
- Badges
- Science method