Major problem with Western blotting - (Aug/11/2004 )

For some weeks we experience a major problem with Western blotting in our lab. When a PVDF-membrane is develop a second time, we couldn´t get any signal and the membrane is destroyed for any further development. This problem is independent of detected proteins and workers. We changed every possible substance but we didn´t solved the problem.

Here is a list of what we changed:
- we used different PVDF-membranes: 0.45µm and 0.2µm
- we used different primary antibodies
- we used different secondary antibodies
- we used different chemiluminescence
- we used different buffers (TBS and PBS, pH is ok)
- we used two different lot nr. of Pierce Stripping Buffer (the problem is still there when we didn´t use any stripping buffer - when developing a membrane a second time without any stripping, we experienced the same problem)
- we used different blocking substances
- we used different films

It´s very strange that the primary development is ok but it´s not possible to develop a second time. We had no problems with this for years, we could strip a membrane up to ten times and we experienced protein loss or anything. When staining the membrane with Ponceau S after this failed second development, we noticed no change - the protein ladder looks normal.

I have run into this problem now. Did you ever solve this problem? If so, how? the strange thing is that, others do not seem to have this problem.
Please help
Ranjani

I dunno, but everyone I've spoken to who uses PVDF eventually runs into
major problem.