ECL
GE Healthcare’s ECL Western Blotting Detection Reagents come in two bottles, a separate reagent in each bottle. For use, equal parts of each reagent are mixed, applied directly to the membrane, and incubated for 1 minute. Excess reagent is drained off by dabbing the corner of the membrane on tissue, and then the blot is covered with plastic and exposed to film. Overall, the reagent kit is good for detecting proteins that are expressed at adequate levels, meaning that a prominent band is present for the protein of interest with loading 10-20 ug of total protein lysate to the gel and a short exposure to film (under 2 minutes).
Blots treated with GE Healthcare’s ECL may show dark background patches depending on the antibody and blocking conditions; however, this is true with any ECL reagent. I have used this ECL on Western blots performed with primary antibodies from BD Transduction, Santa Cruz, Upstate, Sigma, and custom made monoclonal and polyclonal antibodies from Rockland and Abgent, and secondary antibodies from GE Healthcare and Bio-Rad. Regardless of the antibodies used, I have had much success using this ECL. I have had to optimize the primary antibody concentrations for a few blots to reduce background, but only because the concentrations recommended by the vendors were too high for my samples. Also, I get the best results with this ECL when I block my blots (on Hybond ECL nitrocellulose membranes) with 1% BSA and 1% Bio-Rad powdered milk in 0.1% PBS-Tween for either 1 hour at 25°C or overnight at 4°C.
On occasion, I have performed Western blots for target proteins that are expressed at very low levels in my samples, and in these cases, the regular ECL hasn’t been able to robustly detect the proteins. One example of this is when I performed a Rac1-GTP pull-down assay using PAK-PBD beads and then performed a Western for Rac1. In this experiment, the level of Rac1-GTP was very low, and I needed a stronger reagent to detect the protein. So I decided to try GE Healthcare’s ECL Plus.
ECL plus
ECL Plus is good for detecting extremely low levels of a protein. GE Healthcare states that ECL Plus will have 4 to 20 fold greater sensitivity over regular ECL, and that the reagents are optimized for use with PVDF membranes. I have used the ECL Plus for my Rac1-GTP blots (on nitrocellulose) and have produced clear results, strong bands, and minimal background.
Problems can arise though if there is background on the blot using regular ECL, because these dark patches will only be darker and larger with ECL Plus. So optimization of blocking, antibody concentrations, and washing might be needed to minimize background. Also, antibodies will commonly produce a non-specific band or two, and if one of these prominent bands is near your band of interest, ECL Plus might make the non-specific band so bright that your band of interest is covered in its shadow. A simple way around this is to cut off the part of the blot containing the non-specific band, however, this isn’t always possible to do.
Graduate Student
Division of Cardiology
Emory University