About which kit are you speaking?

"Enhanced chemiluminescence (ECL) describes the phenomenon of the light output increase in the reaction of oxidation of luminol catalyzed by horseradish peroxidase in the presence of certain phenolic compounds."

http://www.chem.msu.su/eng/journals/vmgu/00add/109.pdf

So in theory the "dark bottle" contain the luminol, sensitive to light but still stable 8 to 12h.

http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/a8511pis.Par.0001.File.tmp/a8511pis.pdf

I would exclude the ECL problem....

Maybe something else is not working:

Do you have protein on your blot (use amido black staining for example)?

Is your antibody fresh?

Did you put the right secondary antibody?

Is the detecting system working (system able to detect any phosphorescence?)

Good luck!

Best

Agree with Casadei, We always expose the ECL mixture on the blot in the normal room (light); and only for the development of X-ray film you need dark!!

Cross check your method again, you may find something interesting :)

It does not matter with short time exposing to light. There might be some other problem but not ECL, just make sure that you prepare fresh mixture of ECL 1:1 each time required.

I would say that a short exposure of the ECL reaction mix to light will not affect the ability of the technique.

As Casadei already mentioned maybe something else is going wrong.

Could be a wrong secondary antibody, or a primary antibody which has been used extensively.

Also If working with X-ray films there is a possibility the films are accidentally exposed to light and therefore useless.

Hope the experiment works out for you!

Well, thank you very much all of you. For sure, INDEED the ECL is ok. I found out today the problem was with the antibodies. I changed both primary and secondary antibodies and it worked out. Thanks again for the confidence your responses gave me!

(I was too convinced that the antibodies were ok, because I have used them before & the kit says I could recycle about 20 times. I recycled only 2 times, & the problem started on the 3rd attempt)

Interesting that your antibody did not work after 3 usage if before was using after 20.

Could be that you did not block well your membrane?

Casadei, Yes, it was an interesting observation. But I think it has to do with the diluted antibody staying too long at 4 C, as opposed to not blocking well. A bit of some time passed from the 2nd usage to the 3rd.

Well, its the information on the kit that talks about recycling 20 times (We have never reached that number).

Thanx again.

Do you mean that antibody was stored diluted too long? If you are using some reagent like sodium azide it should not be a problem matter.

Best regards

The addition of sodium azide can indeed help the antibody to be preserved for more time at 4oC. I think that you can also freeze the antibody mix. I have done so and reused the antibody and it worked. Although I am not certain whether the antibody will have the same affinity after a long period of time. In general it is not advised to freeze-thaw the antibodies lots of times.

I have experienced this before. Assuming everything else (antibody, etc) is alright, I would try a new batch of ECL. It is quick and might save you from troubleshooting this further. All the best.

I also encountered such a problem, and for me it was definitely not the ECL, but rather some protein degradation in the samples. As for antibodies, I sometimes freeze working antibody dilutions (without preservative) and re-use them. It all depends on the antibody's quality, some work for me perfectly well upon e.g. 4 times thawing, others don't.

Easy way to check if the ECL is OK -mix a small amount of each together, then add some of your secondary antibody in the dark room. You should see a flash of light in the tube, just like a firefly.

That is interesting the secondary antibody directly in ECL.

Now I know what to do if the light switch off on the lab in the middle of the night!!!

Sandra, I agree that is an easy way to check if ECL or secondary Ab is ok. Would you, or anyone, suggest how to check/test if the primary Ab is the one with the problem?

The only way I know is to compare it with a primary Ab which is known to work (that you asked to somebody else in your lab/university but this is not always possible) on a positive control. Of course, you have to be sure that secondary and ECL are working. If there is another method, I'd like to know it as well.

The problem keeps taking new twists! Now, how do you purify the antibody if it has been contaminated? All evidence point to the fact that primary antibody is not binding the protein and someone suggested it may have been contaminated. Any remedy?? The protein of interest is phosphorylated.

Using the new and fresh ECL kit is easy way. Did you try to use ECL prime (new version)?

When testing out the primary antibody as a positive control, should you dilute in 5% milk and TBST and then add the secondary antibody in the same solution?