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Example of uniformly high Western blot background.

Have you ever developed your Western blot only to find that you have a really high background? Nothing is more disappointing then being excited about seeing a new result and literally not being able to see it because it is covered by a big black box. There are several causes of high uniform background. We discussed these in depth in our October monthly online content. Below are a few pointers to help you start to get rid of the big black box.

Don’t waste your antibodies

One of the most frequent causes for high uniform background is using antibodies at a concentration that is too high.Take the time to titrate your antibodies. If you are titrating one antibody, you can do it by running duplicate samples on your gel, cutting the blot into strips and using a different concentration of antibody for each strip. If you need to titrate both primary and secondary antibodies, make a quick dot blot and titrate in a checkerboard like fashion.

Don’t let your block block your results

The blocking step is critical for preventing non-specific reactions and inadequate blocking can result in a high uniform blocking. However, blocking reagents can also be incompatible with certain antibodies leading to a black blot.

Wash that black right out of your blot

Sufficient washing can make a world of difference in reducing high background. Washing removes excess antibody that is not bound specifically to a protein. Wash your blot with at least 3 changes of wash buffer, incubating for 5 minutes at room temperature for each wash. Agitate the blot while washing and don’t skimp on the amount of wash buffer you use. A greater volume of wash buffer will wash the blot more efficiently.

Another way to improve the wash step is to increase the stringency of your wash buffer by adding detergent that will help disrupt non-specific interactions. 0.05% Tween-20 is commonly added to wash buffer. 0.05% NP-40 can also be tried for higher stringency.

Don’t dry out your membrane

Always be careful to keep your membrane wet and completely covered with liquid during all steps.If you blot your membrane to remove detection reagents, make sure you immediately wrap the blot in plastic wrap before it dries out.

High background can be very frustrating to deal with. But if you take your time to determine which step is causing you the problem, you will be able to optimize the conditions and produce publication quality results! If you are having trouble with uneven or splotchy background, read Western Blot Rorschach.

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