The band of beta-actin is just like a continuous line.
Parameter：
Load protein:20ug
Run 80V in stack gel 120V in separate gel
Wet transfer constant 100V for 1h
Use BSA to block for 1h
Primary antibody from CST Incubate in 4o room over night
Second antibody incubate in room temperature for 1h
ECL Reagent from Bio-rad
To my mind, this may be caused by salt/not-SDS detergents in the electrophoresis samples or by a too high concentration of bis-acrylamide in the gels. So, you may try to get rid of salt while making cells extract, or make bis-acrylamide concentration a bit lower (the size of pores in the gel depends on the concentration non-linearly, but U-shaped).
Another variant - excessive heating of the gel during the electrophoresis, but the gel should be significantly warmer, than the room temperature, so it is less possible. How much does the run usually takes? Do you take the pictures of your ECL signal with automatic or manual focus adjustment?
In my experience, loading too much protein or exposure too much time when developing films usually lead to melting line. You can try
1) loading less protein lysates, i.e., 15 ug;
2) exposure less time (2-5 seconds) or use two films simultaneously, and use the second one.
Hope it will work for you!
Take care while preparing gel. Take comb gently, and put the gel in buffer immediately.
Hi,Darya Meshalkina
Thanks for your answer.
I use RIPA lysis buffer from thermo and the gel is 12%
The electrophoresis is between 1.5h and 2h
I use bio-rad imager to receive ECL signal automatically.
Hi,Han Zhang
Thanks for your suggestion.
I think you're right.
Could you explain why use the two films simultaneously ?
Hi,Goodwin G. Jinesh
Do you mean that when electrophoresis is done, the gel should be put in transfer buffer as soon as possible?
- Badges
- Science method