The major question would be why do you need to do this?

But the answer is probably because the HRP enzyme is "burnt out" by first reaction and so the signal is lost. Could be that you are using the secondary antibody at too dilute a concentration - this might explain why you are able to get the signal back by reincubation with the secondary. You should be saturating the binding sites in the fist step and so repeating the secondary antibody should not work as all sites are bound (unless you are stripping it off in the steps you are using - in which case the primary antibody should also be lost to some degree.

Hope that helps.

Hello Christine,

so far I haven't had this problem.

I am using the "enhanced luminescence detection kit" from PIERCE BIOTECHNOLOGIES / Thermo Scientific and could re-apply newly mixed ECL solution without problems.

Maybe you have to change the product or check the best before date of solutions.

Hopefully this is somehow helpful.

Good luck!

Your ECL reagent will include some hydrogen peroxide, and this can be very hard on the antibodies and the HRP itself; so incubating in ECL substrate will inevitably degrade your signal at least somewhat. If you're going to re-develop a blot, I recommend you strip it and reprobe with both primary and secondary antibodies.

The major question would be why do you need to do this?

But the answer is probably because the HRP enzyme is "burnt out" by first reaction and so the signal is lost. Could be that you are using the secondary antibody at too dilute a concentration - this might explain why you are able to get the signal back by reincubation with the secondary. You should be saturating the binding sites in the fist step and so repeating the secondary antibody should not work as all sites are bound (unless you are stripping it off in the steps you are using - in which case the primary antibody should also be lost to some degree.

Hope that helps.

I was just going to type a very similar response to that of Richard Daniel above. If your HRP is fully saturated in the first attempt, then simply adding more ECL solution will not produce signal again. By adding fresh secondary antibody, you are competing with the old secondary already bound and therefore fresh HRP-conjugated antibody will bind to your primary antibody and interact with the ECL reagent, giving you a signal again - although it may not be as strong as the first time. Why do you need to do it twice? The ECL type reactions these days are very good and have very long light emitting times, so there shouldn't really be a need to re-expose, if you are not changing the primary antibody to look at a different protein.

Hola Cristina, Tuve una experiencia igual hace unos años, el caso que tiene tanto sustrato que la reacción de HRP se desarrolla tan rápidamente que no la detectas , por lo tanto añadí unos 10 micro litro de peróxido de hidrógeno para eliminar un poco el sustrato y revelar después de 1 min 2 min y 5 min, o normalizar las cantidades de proteínas (a menos cantidades.). Espero que le he ayudado .

In my opinion it’s better to find the best Xray film exposure time.

I agree with Richard. Especially if you see brown bands develop after the first ECL exposure, free radicals from the reaction will inactivate the peroxidase. Try titrating the secondary (stat with very dilute, maybe 1:100000) until you get optimal exposures at about 30 seconds. You can always incubate with more concentrated secondary if the staining his too light.

Richard Daniel's answer is correct. The HRP enzyme conjugated to the secondary antibody is exhausted during the incubation in the ECL reagent. However, there are substrates (Pierce's SuperSignal comes to mind) which are capable to produce a signal lasting for several hours with a gradual decrease in intensity. This feature can be used to re-expose your membrane later without the additional incubation with secondary antibody.

Pierce's West-femto ECL gives a very strong signal as well (stronger than most others). 1:20,000 to 1:50,000 dilution of secondary works well; and you can leave the membrane submerged in about 1 mm height of the ECL reagent while taking pictures. E.g. begin taking pictures, then submerge the membrane. In other words, immediately start taking pictures even before submersion to catch every second of the process, and snap away for 5 to 10 minutes. So, in essence, we digitally 'film' the development from slightly before submersion all the way through burn-out (if that occurs) and then choose the best frame in the end, using about 10-second development time per frame. The pictures are always very clear - even with the membrane submerged. Your problem of having to re-incubate with secondary to regain signal sounds like there are other complexities going on though - as others have noted.

But film the whole event... each time ... from before the ECL reaction starts, to the 'end.' It simply doesn't make any sense to lose data when it is being generated if it is already possible to film it before it even starts... and continue filming onward from there... for a suitable/informative length of time.

I should clarify that what I meant by "film" is digitally capture the process picture by picture 5 or 10s apart (no film involved - just a good camera and software for photoimaging) in an enclosed photo-imager unit (in the event that something like a phosphoImager or a LiCor Odyssey is not readily available, etc.). Sorry for the confusion.

I have faced similar problem during my western experiments. In such cases, i tried first with Pierce West PICO to get a signal and if i get a weak signal i washed the membrane twice with TBS and reincubate immediately with Pierce West FEMTO (but you might get background).

If you can strip and reprobe your membrane then i would recommend you to do that.