I have so far tried to use a the Restore Western Blot Stripping Buffer to remove primary and secondary antibodies from a PVDF membrane, but have struggled to remove all antibody staining effectively, despite following the buffer instructions, and then increasing incubation of the membrane in the buffer for up to 6 hours.
Hi Elloise,
I previously compared the performance of different antibody stripping buffers, and the one with SDS formulation (containing β-mercaptoethanol) worked relatively well — though it does have a strong odor. This type of buffer is typically used together with heating, so you could give it a try. You can also refer to this paper for the formulation: https://pmc.ncbi.nlm.nih.gov/articles/PMC2679975/pdf/nihms103729.pdf.
In general, these stripping buffers may not be able to completely denature the protein during stripping. If the target proteins you are detecting have different molecular weights, the stripping tends to be more effective — you can use the band size to determine if the previous protein has been successfully removed.
If you use HRP-conjugated secondaries, you can use 30% hydrogen peroxide for 5-10 minutes in an oven at 37ºC. Then wash well with distilled water and you can do the next labeling, preferably alternating the hosts. This stripping saturates the HRP, but it also changes the pH, causing the binding of the antibodies to be weakened. It usually works well, but it may have some problems removing signal from very abundant proteins, such as actin.
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