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
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 µl (38%) per 5 ml ECL solution); however, I do not habe an explanation for your observation...
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
may be someone contaminate the ECL reagents...buy a new set of ECL reagents.
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.