I was testing some samples and my antibodies on a western blot protocol that has worked fine other times. When revealing a membrane on a BioRad Chemidoc this is what showed up. The picture is a mergre of the chemiluminescence and and colorimetric analysis, but for sure comes from the chemiluminescence shoot. What do you think could cause this?
This issue may be attributed to various factors. Firstly, it is crucial to select an appropriate blocking buffer and extend the incubation period accordingly. Secondly, increasing either the antibody concentration or incubation duration could prove beneficial. Lastly, ensuring thorough membrane washing is imperative.
This could be because of few reasons:
1. Buffer or antibody contamination.
2. Concentration of the antibody used.
3. Dirty membrane
4. Insufficient or incomplete blocking of the membrane
5. Or may be too long exposure time
6. Excessive substrate incubation
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