Hi, I have recently performed a WB and got a very strange result when scanning my blot. There is a dotted pattern at the top and the bottom of my blots (see picture).
I have used a rotating tube to incubate my blots with antibodies, made sure they were not overlapping and washed them wit TBS-T and TBS before scanning with ECL substrates.
I can rule out a problem in the transfer, because another blot that was transfered at the same time and that was incubated with a different antibody, and a shaker did not show this abnormal pattern.
Does someone have any idea how this is possible?
Dear Sander,
This could the membrane dried, and the salt crystals destroyed it causing non-specyfic binding. Welcome to add your picture to my collection;)
https://www.researchgate.net/post/Can_you_show_the_picture_of_your_worst_Western_blot_and_explain_the_reasons_for_this_disaster_please
Best - jan
Dear Sander,
This could the membrane dried, and the salt crystals destroyed it causing non-specyfic binding. Welcome to add your picture to my collection;)
https://www.researchgate.net/post/Can_you_show_the_picture_of_your_worst_Western_blot_and_explain_the_reasons_for_this_disaster_please
Best - jan
I do agree with Jan, most probably it is some defect in membrane, eithzer before transfer, or drying during antibody incubation.
Hi,
It may be due to residual lumps of milk powder if you made up your antibody in milk/TBS-T. Ensure to filter the milk/TBS-T mix prior to the addition of antibody. Ensure to also frequently change the TBS wash buffer to remove background antibody binding ie. its better to do 7x 3mins washes rather than 3x 5mins washes. Hope this helps.
I agree it may concern your transfer. I had sth similar at the top of the blot and it was in the place where my gel was too sticky to the mambrane. Or maybe the salt cristals.
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