Dear Dr. Denny Kollareth

The protocol that you have shared could have potential negative effects on data quality. The process of stripping and re-probing can take a considerable amount of time and reagents. Moreover, the membrane stripping procedure may remove sample proteins from the membrane. Therefore, quantification of phosphoproteins following membrane stripping would not be ideal.

I would suggest that the above drawbacks from the traditional chemiluminescent method of detection could be overcome by using fluorescent western blotting which will allow you to detect both versions of the protein (multiplexing) on the same membrane.

Multiplexing your western blot typically require antibodies against the total and phosphorylated protein raised in two different species. Fluorescent secondary antibodies bearing two different fluorophores (with non-overlapping spectra) can be used allowing for multiplexing which means both phosphorylated and non-phosphorylated species can be detected simultaneously.

Another important aspect is to select antibodies which are highly specific for the phosphorylated protein state only. If the antibody is not specific, this can result in simultaneous detection of total protein and confound the interpretation of your results.

Other factors that you should consider while detecting phosphoproteins are:

1. The phosphoprotein must be kept intact. For this reason, protease and phosphatase inhibitor cocktails must be added to cell lysis buffer prior to use. These will help prevent proteins from being degraded and dephosphorylated. Keep the lysate always on ice.

2. You should avoid using milk for blocking the membrane because milk contains casein, an abundant phosphoprotein that can cause high background. Use BSA instead.

3. You should avoid using phosphate-based buffers like PBS as it can interact with anti-phospho antibodies, affecting the binding of the antibody to the target protein. Use Tris-based buffers, for instance, Tris-buffered saline with Tween-20 (TBST) instead.

4. Use proper controls.

Regards,

Malcolm Nobre

An alternative method for detection of both phospho and total levels of a protein by Western blotting is to use a phos-tag gel. The phosphorylated protein will migrate more slowly through the gel, and using the antibody that will detect the total protein should be sufficient. See this: https://labchem-wako.fujifilm.com/us/category/00908.html

I agree with both answers above. Meanwhile I have used the blot-strip-reblot method with decent success (consistency, no crossover, etc.).

I use CST/Thermo antibodies at 1:1000 dilution and CST's protocol: block, incubate overnight, then incubate with 2' for 1 hour and image. Then I use Abcam's stripping buffer (https://www.abcam.com/protocols/western-blot-membrane-stripping-for-restaining-protocol), prepared fresh every time, to strip the blot and repeat the process beginning with blocking.

Thanks all for the comments. I have a concern regarding using fluorescent detection. As Malcolm Nobre wrote above, if we use antibody for phosho and total protein together, will there be a competition between the antibodies? The phospho antibody binds only to the phospho protein, but the total protein antibody will bind both phospho and non phosphorylated form. So, when we add the antibodies together, total Ab will also compete for binding to the phospho protein and I am concerned if this can result in both phospho and non phospho antibodies competing for binding. Please let me know your thoughts about it.

You can look for the epitopes on the product page / ask for technical assistance with binding sites. Usually, they will be pretty far apart. If you need, you can use a protein model to see accurately where the sites are and whether they share the same occupancy. We frequently use both phosphor and total antibodies in immunofluorescence imaging, both should fluoresce.

Leran Mao Thank you so much.