western blot ECL vs fluorescence - (May/03/2006 )
I'm trying to quantify viral proteins. I don't really need to determine the exact amount of proteins but I need to be able to compare different samples. I mean, I want to be able to say that in one sample I have more protein than in other in a reliable way. I will sacn my blot and compare the bands for the protein. I have been told that ECL chemiluminiscence method is not as reliable as fluorescence. I can use both and scann after in a typhoon scanner. Does anyone know if it is like this? If fluorescence is better to quantify than ECL? Thank you very much.
with ECL you can quick saturate the signal, you have to be careful.
I would say fluorescence is better, but I never compared myself.
I use chemifluorescence detection with GE's ECL Plus reagents and visualise on a STORM 840. It works fantastically and is far more flexible and hence more accurate than chemiluminescence.
If you can do it then it is the better option. No need for a dark room and you can quantitate your results directly from the scanned image. You need to consider possible problems though. One being whether you have the right laser on your phosphorimager and another is using low-fluorescence PVDF. There are some other little issues.