I use Pierce SuperSignal West Pico Chemiluminescent substrate for many years, it's far more sensitive than the majority of conventional ECL substrates and the WB conditions may be easily optimized to eliminate the exessive background. When we tested it side-by-side with Pierce ECL under conditions optimized for West Pico, we failed to receive ANY signal using Pierce ECL.

There are even more sensitive kits in Pierce lineage - namely, SuperSignal West Dura Extended Duration Substrate and SuperSignal West Femto Maximum Sensitivity Substrate. I tested the first one, it is even more sensitive than West Pico but requires really extensive optimization to counteract background, band saturation issues and detection of non-specific protein-Ab stickiness having nothing with specific binding. We occasionally use West Dura as a "salvage therapy" for blots with no or extremely weak signals. I have no experience with West Femto (that is even more sensitive) since I really scared to accidentally evoke the devil using this kit :)

I've tested several side by side. Technically my experiments merely measured signal intensity, not sensitivity per se. In my experience: 

INTENSITY:

HIGH  --------------------------------------------- LOW

GE ECL Prime >> Pierce ECL > GE ECL Start

ECL prime give very intense signal, but it can cause background problems and may require modifications to your blotting procedure. I use it for a few very weak monoclonal antibodies, but it required very thorough washing to keep the background down. They do sell a special blocking reagent, which may help slightly.

I use Pierce SuperSignal West Pico Chemiluminescent substrate for many years, it's far more sensitive than the majority of conventional ECL substrates and the WB conditions may be easily optimized to eliminate the exessive background. When we tested it side-by-side with Pierce ECL under conditions optimized for West Pico, we failed to receive ANY signal using Pierce ECL.

There are even more sensitive kits in Pierce lineage - namely, SuperSignal West Dura Extended Duration Substrate and SuperSignal West Femto Maximum Sensitivity Substrate. I tested the first one, it is even more sensitive than West Pico but requires really extensive optimization to counteract background, band saturation issues and detection of non-specific protein-Ab stickiness having nothing with specific binding. We occasionally use West Dura as a "salvage therapy" for blots with no or extremely weak signals. I have no experience with West Femto (that is even more sensitive) since I really scared to accidentally evoke the devil using this kit :)

Hi Stephanie,

Yes, it is a good idea to confirm the transfer. In addition of staining the post-transfer gel, you can also stain the membrane itself with Ponceau S solution or Pierce sells an excellent kit for that purpose called "Reversible Protein Stain Kit for Nitrocellulose Membranes". How are your Western markers showing up on the blots? If they look good, then I guess it is not the problem of ECL sensitivity but of low abundance of the protein of interest or even suboptimal concentrations of primary and secondary antibodies.

Hi Stephanie,

I have good results with Pierce ECL Western Blotting (#32106, Thermo Scientific).

However, I always stain the membrane with Ponceau S to confirm protein load. It takes 20 min to stain and wash and you can have a pretty good idea of your transfer and protein amount. If you are using PVDF membranes, the stain will be low but still you should be able to detect something ;)

Best!

https://www.thermofisher.com/order/catalog/product/34095

Greetings.

Based on my experience and in agreement with previous speech I suggest SuperSignal™ West Femto Maximum Sensitivity Substrate. Now I use myECL™ Imager to detection the presence of proteins and with this ECL I have no problem.

Best regards

Hello Stephanie 

I use Amersham ECL Prime Western Blotting Detection Reagent.

Product code : RPN2232

Amersham ECL prime Western Blotting detection reagent from GE Healthcare provides high sensitivity for chemiluminescent detection of immobilized specific antigens labeled with Horseradish Peroxidase (HRP).

Its has better sensitivity. You can try it.

Pretty much everyone speaks about the background issues related to higher sensitivity ECL reagents. The West Femto Max kit, being the most sensitive one on the market, receives most of the complaints in this regard. When you follow the instructions given by Thermo Fisher, that is, after incubating membrane in reagent A:B (1:1) for 5 minutes, you will most likely end up with a black blot. To get rid of the background, you really need to dilute the antibodies to the extent where you don't detect anything due to too much antibody dilution.

I have crucial suggestions who are willing to get the best out of the West Femto Max kit. First of all, forget about the commercial protocol. Keep using the same primary antibody dilution as you'd use with a regular enhanced ECL kit, or you could dilute twice more. Then use HRP-conjugated secondary antibody in the 1:10.000 - 1:20.000 range (which is roughly 10 times more diluted than what you'd use with a regular enhanced ECL kit). After thorough washes, mix ice-cold reagent A and B, keep it ice-cold, and then add it onto the membranes while the membranes sit in a dish on the tray of the imager. The membranes will stay submerged in the solution (if you have only one membrane piece, pipette a small volume of the reagent mix directly onto the membrane to save on the reagents). Then immediately start imaging in signal accumulation mode with short intervals. You will get very clean and strong signal within seconds or a minute, after which the blot will turn black. So, do NOT follow the stupid "5 min-incubation" step and the excessive antibody dilutions described in the commercial protocol.