Blot Once, Probe Twice - when such approach can be beneficial?

It may happen that sample source is limited and therefore using a membrane twice and probing with another antibody appears to be a good solution. 

As depleting the membrane of antibodies for a second round of detection may involve rather harsh protocols, it is recommended to consider abundancy of a protein which is to be visualized using specific antibodies. It can be checked in Protein Abundance database. The reason for it is a possibility to strip not only antibodies from a membrane, but also protein of interest, which will contribute to less intensive signal and in turn can lead to increased background. 

If we intend to use the same set of samples for detection with several antibodies,
instead of stripping a membrane, two or three primary antibodies can be applied simultanously as described here.

Stripping should not be applied when: 

There is no antibody binding following the stripping, this is not a method to apply. 

Quantitative work is to be conducted. 

Stripping Methods:

• This is a common and relatively harsh method that involves using a buffer containing SDS and β-mercaptoethanol (or DTT) at an elevated temperature (50-70°C). 

• The buffer disrupts antibody-antigen interactions and denatures the antibodies, allowing them to be washed away. 

• • A thorough wash with TBST or PBS is crucial after stripping to remove any residual stripping buffer components.

• This method utilizes a glycine buffer at pH 2.0 with SDS to remove antibodies. 

• The low pH disrupts antibody binding, and the SDS helps solubilize the antibodies. 

• This method is considered less harsh than the heat and detergent method and may be more suitable for some antibodies.