I did a western blot to detect my target protein (42 kD - 6th ladder region) then, after removing the ultra ECL off by doing a few quick rinses, I incubated my membrane with a-tubulin (5th ladder region) overnight at 4C (1:1000). Washed with 1X TBST 3 x 10 min. Secondary antibody was 1:5000 for 1 hour at RT then another 3 x 10 min wash. Imaged with ultra ECL again. Exposure time was 1s.
Hi Gabrielle,
this looks like so called ghost bands to me and this might indeed indicate a very strong signal - for this there can be various reason.
What is happening if you have a very strong signal is that the intense localized signal quickly exhausts the chemiluminescence substrate, leading to no light production during development.
I would check out this site by Biorad: https://www.bio-rad.com/de-de/applications-technologies/western-blot-doctor-protein-band-appearance-problems?ID=MIW4Q8C4S#ghostprotein
as there give some advice here what to do (but ignore what is written directly beside the picture they provide about non-specific bands - I think it does not really fit to the issue, but their advice regarding ghost bands is quite good).
I hope this helps
looks like overexposure, if you already flash exposed the membrane you can try to load less protein to your blot.
Gabrielle Smith , your α-tubulin Western blot signal is indeed too strong.
Possible causes:
Recommended optimization methods:
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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