I'm trying to figure out if the HRP-conjugated antibody in my Western blot is any good. It is a few years old and has been stored according to directions, however nothing turned up on my blot when using chemiluminescent and chromogenic techniques.
What are some ways you have found to find if the antibody is still good? I've heard of using the ECL reagents or DAB with H2O2, but I'm unsure what dilution the Ab would be best when doing either of these.
Any advice would be extremely helpful!
Thank you
In that case you shoud use a primary antibody for an established and/or highly expressed control protein (e.g. GAPDH). when using the corresponding secondary antibody you should get a quite good signal. After that, you could try an other established antibody for proteins expressed in lowe amounts (maybe transcription factors etc.).
Hi Sarah,
If you want to know your old HRP conjugated antibody work correctly, you must do 2 simple experiments.
First you have to make sure that conjugated enzyme works properly: add 1-2µl of HRP-conjugated antibody to 100µl of TMB or DAB solution. If you see the color change, it works fine.
Then do a dot blotting with primary antibody. At the first add 1-2µl of primary antibody directly on PVDF membrane( like a dot). After blocking of the membrane keep on the rest of the steps like Western Blotting. If you dont see any blot after ECL, it means your antibody are expired and you must by a new one.
Good Luck!
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