Problems with western membrane stripping - (Aug/19/2013 )
Hello fellow scientists,
I am a humble beginner in western blot, I tried to strip and reprobe my pvdf membrane with several antibodies but was unsuccessful, the membrane remained blank even after I tried reprobing with 5 different antibodies. Here is the procedure I used.
1. I ran a 10% SDS-Page with my samples
2. Transfer onto a pre-wet PVDF membrane
3. Block with 5% skimmed milk for 1 hour
4. Probed with Beta Actin O/N
5. Developed membrane using HRP-ECL
6. Stripped using a semi-harsh method
Mild stripping buffer, 100 ml
1.5 g glycine
0.1 g SDS
1 ml Tween-20,
Adjust pH to 2.2, top up with water to 100 ml
- incubate membrane in buffer for 10 mins at RT for 2 times, discard
- wash membrane with PBS for 2 times
- wash membrane with TBST for 2 times
7. Blocked with milk again
8. Reprobed with a second antibody.
9. Developed ECL.
Can anyone help me?
So it worked at the B-actin but not for the second one?
If so, do you have a positive control so that you know that antibody is working? Could you try your second antibody first in the place of B-actin and see how you get on?
why re-blocking? this standard stripping should not release polypeptides bound to the membrane directly but antibodies