I am working with histone sub-unit 3 lysine 9 position (H3K9) and looking for particular modifications that are happening there (beta-hydroxybutyrylation and butyrylation). I used H3K9BHB as a primary antibody and then a secondary antibody (H3). Now, I want to use H3K9Bu as another primary antibody. The problem is that for both antibodies, the target is H3K9. In this scenario, when I strip H3K9BHB antibody and then reprobe with H3K9Bu antibody, there is already some signal/band left from previous antibody (H3K9BHB). How can I know that the signal/band I am seeing is from H3K9Bu but not the leftover of H3K9BHB? Shall I use different blots to use these antibodies separately? Is there any technique that can remove the bands of H3K9BHB completely (without affecting the protein in PVDF) so that I can reprobe another antibody targeting the same location?
The protein of my interest is of 17 kDa size. I am currently using NewBlot PVDF stripping buffer from LICOR for 30 minutes for stripping the antibodies.
Hi Sachin,
I'm not sure which method you're currently using for stripping antibodies but there are generally 2 methods: a mild method (which strips by low pH) and a stringent, harsher method (which strips by heat and detergent).
You can look at the following sites for info that I found useful when I initially started stripping my membranes:
- https://www.cytivalifesciences.com/en/us/news-center/stripping-and-reprobing-western-blot-membrane-problems-and-solutions-10001
- https://www.sigmaaldrich.com/ZA/en/technical-documents/protocol/protein-biology/western-blotting/stripping-and-reprobing-western-blotting-membranes
- https://www.thermofisher.com/za/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/stripping-reprobing-western-blots.html
- https://stjohnslabs.com/blog/western-blot-membrane-stripping
As some of these sources mention, you might consider the type of substrate you use to develop your reaction/signal. Some advise not to strip membranes when you've used chromogenic subtrate like TMB. But I use TMB to develop my western blots and I've stripped my membranes with heat and detergent (stringent method) - I haven't had any issues. No loss of protein or issues with subsequent antibody binding, and no faded/partial bands/signal left behind on my membrane after stripping.
It's also very important not to let your membrane dry after stripping. If, for example, you strip today and only plan to reprobe your membrane the following day, leave your stripped membrane overnight in wash buffer. I do this all the time and it's always worked for me.
So, consider (1) the type of substrate you're using, (2) maybe use the harsher/stringent stripping method to try and get rid of as much signal from the first antibody as possible, and (3) making sure your membrane doesn't dry out after stripping.
Assuming that you are using fluorescent detection and you can get primary antibodies which are raised in two different species, a better way would be to simultaneously probe with both primary antibodies at once and see the relative intensity of the signals for both antibodies using different fluorescently tagged secondary antibodies.
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