Hi Rajni,
we have found that one of the simplest stripping methods for nitrocellulose is the following:
1) Wash membrane in TBS-T for 5 min
2) Add 0.2M NaOH for 1-2 min and wash with agitation. 2 mins is usually more than enough but can go 5 mins if necessary.
3) Wash membrane with lots DI water, changing every 2 minutes, do 5changes at least to ensure all NaOH is gone.
The NaOH kills the HRP as well as removing some of the antibodies
Note that Rabbit polyclonal antibodies are sometimes harder to remove than mouse monoclonal antibodies.
Then reblock with skim milk or BSA as you would usually and then you can reprobe.
Remember, always start with your weakest antibody and then reprobe with those that give stronger signals, ending with loading control (i.e. beta-actin).
You can use the same stripping system for PVDF membranes as well.
Good luck!
Charles de Bock
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