What is a Western blot?
Western blotting is the process of transferring proteins from a gel onto a membrane to identify proteins of interest. The membrane is treated with antibodies that bind epitopes on the protein of interest. After further treatment with a secondary antibody, specific proteins in the sample can be visualized using detection techniques such as the Enhanced Chemiluminescence (ECL) method.
The Western blot is a lengthy procedure, consisting of many incubation and wash steps. To save reagents, samples, and time, use a single membrane to detect multiple proteins of interest and avoid the necessity of running multiple gels.
What is blot stripping and what are its advantages?
Blot stripping is a technique that removes the primary and secondary antibodies attached to a Western blot that has already been used to detect a protein. It enables the blot to be subsequently re-probed with another antibody, allowing the detection of additional proteins of interest on a single blot.
Another advantage of stripping is that the same blot can be used to detect a loading control, negating the possibility of user bias or error in detecting of the relative levels of the protein of interest. Also, the same blot can be used to optimize antibody concentration for the detection of a protein without the need for multiple gel runs and transfers.
How is blot stripping performed?
Stripping is done mainly in two ways – Mild stripping or Harsh stripping. Stripping methods utilize a combination of detergent, reducing agent, heat, and/or low pH that weakens antigen-antibody interactions and allows the antibodies to be washed away. However, these treatments can also interfere with protein-membrane interactions. Therefore, to preserve membrane-bound sample proteins, one should always start with mild conditions.
Considerations BEFORE stripping a membrane:
- Choice of membrane – The hydrophobic nature of polyvinylidene difluoride (PVDF) membranes offers higher binding capacity and better retention of adsorbed proteins as compared to nitrocellulose membranes. PVDF membranes are also less fragile, making them a better choice when blots must be stripped and re-probed.
- Choice of detection method – Stripping must be performed on membranes where protein detection was done using either ECL or fluorescent methods because the visible stains left on the membrane by colorimetric methods cannot be removed by stripping. Also, as the streptavidin-biotin bond is very strong and can be difficult to dissociate for re-probing, it is better to avoid avidin conjugated probes.
- Choice of stripping method – To avoid loss of sample, always start with mild conditions and use harsher conditions as required to break up stronger antigen-antibody interactions.
- Choice of the sequence of protein detection – Always prioritize the detection of low abundance proteins and the use of low-affinity antibodies, as with each successive stripping some antigen loss is inevitable.
Considerations AFTER stripping a membrane:
- Post-stripping washes – It is extremely important to wash the stripped membrane excessively to remove any traces of stripping buffer components before re-probing. Stripping buffers contain reducing agents such as β-Mercaptoethanol, traces of which can denature any antibody that is used after stripping.
- Blocking – It is necessary to re-block the membrane after stripping to prevent the non-specific binding of antibodies to the membrane. Blocking buffers vary in composition from complex protein mixtures in milk-based buffers to single-protein formulations as in casein and BSA-based buffers. Non-mammal and non-animal protein blocking buffers are also available. Components in blocking buffers can interfere with antibody binding in some cases, so choose a blocking buffer appropriate for your application.