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Problems wth self-made luminol - (Aug/07/2008 )

I have problems with self-made luminol, I made it from luminol (Sigma) and p-coumaric acid (Sigma) by this protocol: 1 ml luminol (250mM in DMSO) + 0.44 ml p-coumaric acid (90 mM in DMSO), 10 ml 1M Tris-HCl pH 8,5, filled with destilated water to final vol 100 ml. This is so called Solution A, while Solution B is 12,2 microl 30% H2O2, 2 ml 1M Tris-HCl pH 8,5 and up to 20 ml dd water. For ECL I use Sol. A: Sol. B= 1:1. I have several problems:
1. After some time I cant get a signal by ECL while it is where good after dying membranes with DAB
2. Some antibodies work fine with ECL while others (with same secondary antibody) after some time dont work
3. Often I dont get signals for samples at the end of membrane while middle works fine?

Thanks for advices

-natasav-

QUOTE (natasav @ Aug 7 2008, 02:07 AM)
I have problems with self-made luminol, I made it from luminol (Sigma) and p-coumaric acid (Sigma) by this protocol: 1 ml luminol (250mM in DMSO) + 0.44 ml p-coumaric acid (90 mM in DMSO), 10 ml 1M Tris-HCl pH 8,5, filled with destilated water to final vol 100 ml. This is so called Solution A, while Solution B is 12,2 microl 30% H2O2, 2 ml 1M Tris-HCl pH 8,5 and up to 20 ml dd water. For ECL I use Sol. A: Sol. B= 1:1. I have several problems:
1. After some time I cant get a signal by ECL while it is where good after dying membranes with DAB
2. Some antibodies work fine with ECL while others (with same secondary antibody) after some time dont work
3. Often I dont get signals for samples at the end of membrane while middle works fine?

Thanks for advices


use 3 solutions: A: luminol, B: p-coumaric acid, C: H2O2;

-The Bearer-

QUOTE (The Bearer @ Aug 7 2008, 06:09 AM)
QUOTE (natasav @ Aug 7 2008, 02:07 AM)
I have problems with self-made luminol, I made it from luminol (Sigma) and p-coumaric acid (Sigma) by this protocol: 1 ml luminol (250mM in DMSO) + 0.44 ml p-coumaric acid (90 mM in DMSO), 10 ml 1M Tris-HCl pH 8,5, filled with destilated water to final vol 100 ml. This is so called Solution A, while Solution B is 12,2 microl 30% H2O2, 2 ml 1M Tris-HCl pH 8,5 and up to 20 ml dd water. For ECL I use Sol. A: Sol. B= 1:1. I have several problems:
1. After some time I cant get a signal by ECL while it is where good after dying membranes with DAB
2. Some antibodies work fine with ECL while others (with same secondary antibody) after some time dont work
3. Often I dont get signals for samples at the end of membrane while middle works fine?

Thanks for advices


use 3 solutions: A: luminol, B: p-coumaric acid, C: H2O2;

Can you explain this: concentration of luminol, coumaric acid, etc.

-natasav-

You have to specifiy if this is an antibody or a luminol problem. Some antibodies will not give you a good signal even if everything is allright with your luminol. You can test the luminol by adding a little bit of secondary ab to the 1:1 working solution (small volume is enough), and see if it is glowing in the dark room. If you get no or only a week signal, then you have to make fresh solutions or change the H202. Use the solutions no longer than one week, better prepare a smaller volume of you Sol. A. 20 ml is fine for one week for each solution normally.

-biomaus-

follow the Bioforum discussions:
http://www.protocol-online.org/forums/inde...8524&hl=ECL

-The Bearer-

QUOTE (biomaus @ Aug 7 2008, 10:59 PM)
You have to specifiy if this is an antibody or a luminol problem. Some antibodies will not give you a good signal even if everything is allright with your luminol. You can test the luminol by adding a little bit of secondary ab to the 1:1 working solution (small volume is enough), and see if it is glowing in the dark room. If you get no or only a week signal, then you have to make fresh solutions or change the H202. Use the solutions no longer than one week, better prepare a smaller volume of you Sol. A. 20 ml is fine for one week for each solution normally.

Ive tried this, and its OK, antibody is not problem. Its seems to me that signal is loosing quickly, how long I should incubate in luminol ( my time is 5 min), should membranes be dried or not (PVDF)? Still I have problem with samples on the end while the midlle give nice signals.

Thnaks for advice

-natasav-

QUOTE (natasav @ Aug 13 2008, 11:16 PM)
QUOTE (biomaus @ Aug 7 2008, 10:59 PM)
You have to specifiy if this is an antibody or a luminol problem. Some antibodies will not give you a good signal even if everything is allright with your luminol. You can test the luminol by adding a little bit of secondary ab to the 1:1 working solution (small volume is enough), and see if it is glowing in the dark room. If you get no or only a week signal, then you have to make fresh solutions or change the H202. Use the solutions no longer than one week, better prepare a smaller volume of you Sol. A. 20 ml is fine for one week for each solution normally.

Ive tried this, and its OK, antibody is not problem. Its seems to me that signal is loosing quickly, how long I should incubate in luminol ( my time is 5 min), should membranes be dried or not (PVDF)? Still I have problem with samples on the end while the midlle give nice signals.

Thnaks for advice

5 min incubation does not make sense; beware, that H202 is co-substrate of HRP but damage it; after ca. 20 min, the reaction is over

-The Bearer-

My incubation time was 3-5 minutes, it takes time for enzymatic reaction to start, but after 10 minutes the reaction slows down and your signal weakens. So, incubate some minutes and move the solution constantly over the membrane. The membrane has to be wet but when you develop let the excess luminol drop off your membrane or you might get high background. The middle / end difference in signal strength sounds to be due to blotting procedure or gel running. Make sure your membrane is packed nicely in the blot and no bubbles are under the sheets. For the gel running you should be careful to not overheat your gel so that you have smiley-effects at the sides.

-biomaus-