ECL Substrate Choices - (Apr/16/2014 )

Hi.  I'm new to chemiluminescence western blots.  In searching for ECL substrates for use with HRP-conjugated secondary antibodies, I'm a little overwhelmed with the choices.  I'm pretty confident that I want a mid-sensitivity substrate to avoid the background issues with the ultra-sensitive substrates but there are so many choices.  Does anyone have favorites?

Thanks!

I use supersignal pico from Pierce.

It depends on the amount of protein you have as a band.

We usualy start with

1- Least sensitive--->  Millipore Luminata Crescendo Western HRP substrate

http://www.millipore.com/userguides.nsf/a73664f9f981af8c852569b9005b4eee/6083afbbc21a8690852576ff004a2c8f/$FILE/00112798.pdf

then, if there is no band of interest, we wash the membrane with T-TBS three times each 30 second. then we use this:

2- More sensitive---> Millipore Luminata Forte Western HRP substrate

http://www.millipore.com/userguides.nsf/a73664f9f981af8c852569b9005b4eee/6083afbbc21a8690852576ff004a2c8f/$FILE/00112798.pdf

then, if there is no band of interest, we wash the membrane with T-TBS three times each 30 second. then we use this:

3- Most sensitive---> SuperSignal West Femto Chemiluminescent Substrate

http://www.piercenet.com/product/supersignal-west-femto-chemiluminescent-substrate

but recently we found that CellSignal ECLs are very good and we are going to use these:

6883 SignalFire™ ECL Reagent

12630 SignalFire™ Plus ECL Reagent

12757 SignalFire™ Elite ECL Reagent

7003 20X LumiGLO^® Reagent and 20X Peroxide