We use Western Breeze kit from Life technologies and also the ECL from GE.

You write that PE kit was good for one protein, and GE healthcare for another (actin), suggests that you use see different kits with different antibodies? You might consider using a tagged MW ladder (dual vue) as a QC for your kits. If you think the problem is with the secondary, Jackson Immunoresearch makes some good HRP-conjugated secondaries. Do you know if all the proteins you want to detect are present at comparable concentrations? You could consider using Biotinylated secondary and amplifying with streptavidin-HRP to increase signal. When all else fails going to back to chromagenic detection to get the antibody dilutions can help speed things up.

I started with our kit PE, replacing with fresh ECL to detect the second protein, actin. When I was not able to detect actin, I borrowed some reagent (GE) from another lab and was able to see actin. I assume that two of the proteins are very abundant, but the third may be in lower abundance to the first two. I have heard that the ECL can be depleted by very strong or abundant proteins. Some people detect actin last as not to burn out the ECL. I am labeling the ladder with streptactin and its detection is variable as well again making me think about the ECL. Thanks for the suggested products, I will check them out. We usually use Santa Cruz secondaries.

We have experienced similar things with westerns on recombinant proteins that we make. Sometimes the ECL reagent when applied to the membrane will be exhausted rapidly. A solution for this is to use less protein. You can confirm by doing Alk Phos, it will turn color too rapidly. Yes, detecting Actin last is helpful as the signal can be very strong and difficult to remove once reacted, if you are looking to reprobe the blot. If you know your antibodies are good and protein is relatively stable, you can try slot blots to work out the detection conditions to speed things up then run the western

Another possibility is that you may be washing away your secondary. You can try not doing a typical stringent wash before adding your ECL. I would suggest a few very quick swishes (3-4 over the sink) before adding the ECL just to see whether you can see your signal. You might also want to consider using more protein.

Bio-Rad have another kit similar to Immunostar, it is Western-C. We usually use it and we have good results.

We use the ECL kit from Pierce. It does work well.

For the ECL I use the detection system Pierce™ ECL Western Blotting Substrate which I purchase from ThermoFisher Scientific. There are different sensitivities.

There is also a beta-actin primary conjagated to HRP from Sigma that requires no secondary, can be fast with a dilutions of 1:25K to 1:50K that works well for my cells.

You might also want to do a dose response with a slot blot to determine the sensitivity and dot blots with differing concentrations of primary and secondary antibodies.

Have you tried using SuperSignal West Pico Chemiluminescent Substrate from Pierce/Thermo? I agree with Kevin that you do need a QC control for your WB, like biotinylated Mr standards. To increase the sensitivity of detection, I would also recommend you use biotinylated primary or secondary antibody, and then neutravidin-HRP (Pierce) instead of streptavidin-HRP. Neutravidin usually gives much lower background than streptavidin.

Dear Amy, the concentration of your primary AB seems to be very low, could be increased 10-20 fold. With the ECL supplier don't care to much, the freshness is more important, than the logo;)