An ARP Customer Service Representative is happy to help!
When your blot comes out looking like a bad x-ray, you’ve got background problems. 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.
Unfortunately, you can never tell if you’ve got, or will have, a background problem with a blot until you’ve developed it.
However, should you be having problems with high background across the blot, there’s a lot you can do to improve things. 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.
Again, 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.
This is especially true of the detection antibodies, which could form aggregates if not prepared and stored properly, or if they are quite old.
If you’ve not blocked your blot properly, then your detection antibody will likely bind anywhere and everywhere. To help fix this you can try:
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.
Milk also contains biotin, so it’s not suitable when using an avidin-biotin based detection system.
If you’re blots not well-washed, then ‘junk’ can be left over that interferes with the detection antibody(s).
Primary or secondary antibody – if the concentration of either of these is too high, background may be boosted. Try:
If you expose your blot for too long with the detection reagents, or equipment, may over expose your blot. Try:
A dry membrane can lead to high background
Note: Some nitrocellulose membranes may interfere with luciferase-based detection systems. Check with the manufacturer.
Filed Under : Antibodies