Problems with western membrane stripping - (Aug/20/2013 )

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?

Thanks!

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