I have been doing some troubleshooting relating to western blot with the help of a friend. We decided to test if our anti-rabbit conjugated HRP secondary antibodies are working properly by preparing both of us preparing a ratio of 1:1 (500uL of our substrate) and adding 1uL of our secondary antibody in a dark room. We both saw that immediate strong blue light was given for both of our antibodies. However, my blue light signal faded much quicker (lasted aroubd 25 to 30 seconds) than my friend's blue light signal (lasted maybe 1 minute or a little less).
What would be the reason for this?
We both have different anti-rabbit secondaries from different companies. We both have different substrate from different companies.
I repeated this with the substrate of my friend (from millipore) and there was strong blue light signal but faded much quicker than with my own substrate (super signal west femto)
At the end, both of our substrate were a dark brown color.
Why are you putting your conjugated antibodies straight into your substrate?
If this is to assess the chemo luminescence it makes no sense
The only way to assess your substrate is to use different secondary antibodies on your NC membrane that you then wash and incubate with substrate on the membrane
If you think the ECL reagent has gone off then you need to assess that in the context of an actual blot. Adding secondary immediately to your substrate will give a signal but will have no bearing on the signal emanating from particular antigens bound by your HRP conjugated antibody on your membrane
The first thing you should try is an antibody to a high copy number protein like actin, gapdh or b tubulin
If that fails to give a signal then you probably require fresh ECL (assuming of course you know that aliquot of antibody works for somebody else
Hi Eduardo,
I have also used this test, it is very easy and very quick. To answer your questions, if you use a very sensitive ECL solution like SuperSignal Femto (in my experience one of the most sensitive ECL's) the enzymatic reaction of the ECL Substrate with the HRP will be extremly quick and the enzyme will "burn away" the substrate near to it very quickly (and then you see nearly no glow in the "darkroom test" because the enzyme is surronded by product and not substrate). To use the super-sensitive substrates like Femto really, I would recommend to dilute the 2° ab at least 1:50 000, better 1: 100 000.
If you both use the same substrate and the performance of the other antibody is better, maybe the HRP on your antibody is a little bit "compromised", the enzymatic reaction of HRP will create radicals which not only attack the substrate but unfortunately also the HRP itself. If you use an aliquot of 2°ab several times, it is possible to "contaminate" the 2°ab with substance which influence the work of the enzyme.
Best,
Carsten
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