is tissue from rat kidney reacting with ECL? - (Jun/07/2006 )
I have experienced the strangest ting when I do western blotting. I found that when when I had ECL directly on a membrane (no primary or secondary antibody was added) there was some reaction with a sample from rat kidney and ECL resulting in a big dark spot in my blot. The ECL only reacted with the samples that were made from tissue from the rat kidney. Other samples like mesenterium and skin tissue samples ( from the same rat) did not give this effect.
Is there anybody who knows if there is anything in the rat kidney that may react with the ECL? I have searched the literature and no one rapports anything special about using ECL on tissue from rat kidney...
Is there a peroxidase present in kidney?
I think so, but isn’t peroxidase present in all tissue?
Did the blot have..like 10 lanes and you could see something silmilar to bands? Or was it just a "big dark blob" that does not really correlate with the lanes loaded? In that case I would suggest some extra washing 
And: if there was indeed high levels of peroxidase in every kind of tissue- wouldn't each protein sample on a membrane show massive luminiscence with ECL?
it would be some peroxydases resistant to reduction?
And: if there was indeed high levels of peroxidase in every kind of tissue- wouldn't each protein sample on a membrane show massive luminiscence with ECL?
There were 9 samples loaded on the gel, and only the samples with the kidney showed the reaction. The rest of the lanes were all blank. No antibodides were added so the ECL should not have anything to react with. Any Peroxidose already present in the sample are denatured with boiling and sample buffer. The reason we tryed to test the ECL on the blot with no antibodies was because we always got some strange bands on all sample no matter what kind of antibody we used…
hello,
don't be so confident that boiling and SDS denaturation destroys every protein. quite a number of proteins (including some peroxidase isoforms) can re-nature on a membrane following western transfer. if these peroxidases are abundant in kidney tissue, it's quite possible that you'll get some reation w/ ECL reagent. even if 99.9% of them are destroyed, that 0.1% remaining will be enough to react w/ substrate molecules. given the sensitivity of ECL, that would be enough to make a mess for you.
2 possible remedies to your problem involve using azide, which covalently modifies proteins. it is a very cheap, effective inhibitor of peroxidases. you could either pre-treat your kidney extracts w/ 1% sodium azide for 30 min or so prior to addition of electrophoresis buffer, or you can treat your membrane w/ 1% azide after western transfer for 1/2 hour. block the membrane as you normally would afterward, and be sure to wash it a few times in large volumes before probing w/ Abs and ECL.
after azide treatment and washes, probe your membrane w/ ECL alone. if you don't see any signal, wash it and then re-probe w/ Abs and ECL like you normally would for chemilluminescence.
Did the blot have..like 10 lanes and you could see something silmilar to bands? Or was it just a "big dark blob" that does not really correlate with the lanes loaded? In that case I would suggest some extra washing
And: if there was indeed high levels of peroxidase in every kind of tissue- wouldn't each protein sample on a membrane show massive luminiscence with ECL?
There were 9 samples loaded on the gel, and only the samples with the kidney showed the reaction. The rest of the lanes were all blank. No antibodides were added so the ECL should not have anything to react with. Any Peroxidose already present in the sample are denatured with boiling and sample buffer. The reason we tryed to test the ECL on the blot with no antibodies was because we always got some strange bands on all sample no matter what kind of antibody we used…
after azide treatment and washes, probe your membrane w/ ECL alone. if you don't see any signal, wash it and then re-probe w/ Abs and ECL like you normally would for chemilluminescence.
Thanks, I’ve already tried to wash the membrane with 1% Sodium Azide and the signal got weaker but not completely gone. Maybe the way to get rid of it is to pre treat the samples as you suggested. The protein I’m searching for is located exactly on the place where the ECL reacts so to be confident on my result I have to get rid of this mess…
Can you use an alkaline phosphatase conjugate as a secondary antibody with the CPD-Star substrate (Tropix)/ another alk phos substrate? This might get around your problem in a simpler way but you would need to buy a new conjugate and a new substrate solution.
All the best,
Ceri
Still another possibility is the use of Infrared Fluorescence Antibodies. (eg: LICOR) If you work in a big department probably some groups do have this facility: http://www.licor.com/bio/Odyssey2/Odyssey1.jsp