Our department has a long history of being a leader in the field of basic and translational cancer biology research. Check with the manufacturer.Welcome to MD Anderson's Cancer Biology department. Note: Some nitrocellulose membranes may interfere with luciferase-based detection systems. Ssome membranes have lower background than others. Generally, nitrocellulose tends to have a lower background than PVDF, but try different membranes.Be sure to always store your membrane in a liquid storage medium at all times.Reducing the exposure time, or detection system incubationsĪ dry membrane can lead to high background.If you expose your blot for too long with the detection reagents, or equipment, may over expose your blot.
You can perform a secondary antibody control, omitting the primary antibody, to check if the secondary is the problem.Use a lower concentration antibody with a lengthened incubation.Optimizing your antibody concentrations.Primary or secondary antibody – if the concentration of either of these is too high, background may be boosted. Increase the amount of Tween-20 to 0.1%, if it is not already.For example, wash 4-5 times for 5-minutes. Ensure enough washing buffer is used to cover the blot.If you’re blots not well-washed, then ‘junk’ can be left over that interferes with the detection antibody(s). Milk also contains biotin, so it’s not suitable when using an avidin-biotin based detection system. Tip: If you’re using a phospho-specific antibody, don’t use casein or dried milk as the blocking reagent, as casein is a phospho-protein, and is present in milk. Titrate your primary and secondary reagents in the same blocking buffer.This can help reduce any cross-reactivity between blocking reagent and antibodies. Add 0.05% Tween-20 to your blocking buffer, if it doesn’t already contain it.Increase the incubation time with your blocking buffer, preferably with gentle agitation.For example, block for 2 hours at room temp, or overnight at +4☌. Increasing the concentration of your blocking reagent, whether casein, non-fat dried milk or BSA.If you’ve not blocked your blot properly, then your detection antibody will likely bind anywhere and everywhere. This is especially true of the detection antibodies, which could form aggregates if not prepared and stored properly, or if they are quite old. There are a lot of possible causes for high background in a western blot, and below we outline some of the main causes, and their solutions.Īgain, in all instances, if you have high background problems and need to repeat your blot, it’s best to start with freshly made buffers and reagents, made according to the manufacturers recommendations, to make sure everything is correctly prepared and there is no contamination of buffers. However, should you be having problems with high background across the blot, there’s a lot you can do to improve things. Unfortunately, you can never tell if you’ve got, or will have, a background problem with a blot until you’ve developed it. It’s the result of your detection antibody, or development reagent, binding to anything and everything else on your blot, losing the signal you’re looking for – the protein you’re interested in – in the shadows of this non-specific binding. When your blot comes out looking like a bad x-ray, you’ve got background problems. Common causes of high background in Western Blotting
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