Sometimes my western blots have very uniform/even background, other times they're very uneven and patchy like in the attached image. Why is this?
I thought my blots were drying out during staining/blocking, so I used large volumes, but still I see uneven background.
I am using pvdf, licor blocking buffer, blocking for ~1 hr at room temperature with light shaking.
A contaminated buffer can easily lead to a high background during a western blot. Most commonly, a contaminated buffer will occur due to the presence of bacteria -- but there can be other contaminants as well. https://info.gbiosciences.com/blog/tips-for-preventing-a-high-background-during-western-blotting
Uneven splotchy background or incomplete bands can occur if the antibody is not evenly distributed over the membrane during incubations. Use enough antibody solution to completely cover the membrane. Agitate the blot on a shaker during incubations. https://advansta.com/wikis/high-background-in-western-blots-causes-and-solutions-2/
Kindly check https://www.bio-rad.com/en-us/applications-technologies/western-blot-doctor-blot-background-problems?ID=MIW4O8ESH
A contaminated buffer can easily lead to a high background during a western blot. Most commonly, a contaminated buffer will occur due to the presence of bacteria -- but there can be other contaminants as well. https://info.gbiosciences.com/blog/tips-for-preventing-a-high-background-during-western-blotting
Gen Gi I don't know what exactly your steps are. Anyway, if the blocking and washing steps of the membrane have been done properly, it can be possible that the shaker did not work properly during the incubation steps. Also, be careful while you pour the reagents on the membrane; the background can happen in that area where you pour reagents directly. Moreover, ensure that you didn't touch the membrane, use forceps.
Good luck
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