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ECL gives me lower sensitivity than chromogenic. Why could it be? - (Mar/13/2007 )

Hi,

I have a problem with ECL detection. It looks like I get lower sensitivity with ECL than what I get with chromogenic (which is surprising, right?).
I first used chromogenic detection with AP-conj secondary Ab. It worked but I got very faint bands. Since I am limited by the amount of sample, I decided to use ECL since it is supposed to be more sensitive. I tried Amersham and Pierce ECL kits (SuperSignal West Femto Maximum Sensitivity Substrate). Surprizingly, the signal intensity even after 30 and 60 minute film exposure is lower than what I get with chromogenic (BCIP/NBT for AP-conj detection). I see no bands after 1 to 10 min exposure times and only after 30-60 min I see very very faint bands.
In my ECL westerns I used ~ 100-50 ng/ml primary antibody (the same concentration as in chromogenc detection) and 1 ng/ml of secondary HRP-conj antibody (as recommended by the manufacture).
Does any one know what might be the reason why ECL gives less sensitivity than the chromogenic detection?

Thanks a bunch.

Arina

-anna122-

QUOTE (anna122 @ Mar 13 2007, 07:29 PM)
Hi,

I have a problem with ECL detection. It looks like I get lower sensitivity with ECL than what I get with chromogenic (which is surprising, right?).
I first used chromogenic detection with AP-conj secondary Ab. It worked but I got very faint bands. Since I am limited by the amount of sample, I decided to use ECL since it is supposed to be more sensitive. I tried Amersham and Pierce ECL kits (SuperSignal West Femto Maximum Sensitivity Substrate). Surprizingly, the signal intensity even after 30 and 60 minute film exposure is lower than what I get with chromogenic (BCIP/NBT for AP-conj detection). I see no bands after 1 to 10 min exposure times and only after 30-60 min I see very very faint bands.
In my ECL westerns I used ~ 100-50 ng/ml primary antibody (the same concentration as in chromogenc detection) and 1 ng/ml of secondary HRP-conj antibody (as recommended by the manufacture).
Does any one know what might be the reason why ECL gives less sensitivity than the chromogenic detection?

Thanks a bunch.

Arina



you can increase sensitivity of ECL by adding og H2O2 (up to 1.5 ┬Ál (38%) per 5 ml ECL solution); however, I do not habe an explanation for your observation...

-The Bearer-

I had a similar problem a while back, so this may be a remote possibility. It took quite some time to figure out that the Na-Azide used in my wash/block buffer to prevent microbe growth, but it also inhibits the peroxidase reaction, which is why you see a product with Alk. phosphatase but not HRP. Hope that helps. Cheers!

-JAH-

QUOTE (JAH @ Mar 13 2007, 07:33 PM)
I had a similar problem a while back, so this may be a remote possibility. It took quite some time to figure out that the Na-Azide used in my wash/block buffer to prevent microbe growth, but it also inhibits the peroxidase reaction, which is why you see a product with Alk. phosphatase but not HRP. Hope that helps. Cheers!


hi Jan,

Thank you for you comment. I have read about the inhibitory effect of NaN3 for peroxidase activity. I did not have any of azide in my buffers. There is no NaN3 in neither secondary nor primary Ab's that I use. There must be something else that inhibits the detection but I can't figure out what it is. I followed Piece protocol to the letter... So I am still looking for the answer.

If someone has other ideas, please let me know. I will be very grateful!

Arina

-anna122-

may be someone contaminate the ECL reagents...buy a new set of ECL reagents.

-Minnie Mouse-

as aimikins points out in another thread (someplace around here), too much secondary can cause ecl to burn out rapidly so it will appear that you don't have the sensitivity.

-mdfenko-