Chemiluminescent Western Detection

Everything you need for chemiluminescent Western blotting

Advansta's WesternBright line of HRP substrates contains a solution for every chemiluminescent Western experiment, from routine blotting to quantitative comparisons, from film detection to CCD imaging. Each substrate has been optimized for high-performance enhanced chemiluminescence detection (Figure 1) for a specific experiment type, ensuring successful, reproducible Western blots for every sample and situation.

Figure 1. The principle of Western blot detection by enhanced chemiluminescence. A primary antibody binds to the protein of interest, which is immobilized on a membrane support. The location of the primary antibody is detected using a secondary antibody directed towards the primary antibody, and conjugated to horseradish peroxide (HRP). An HRP substrate is applied to the blot, and light is generated as a product of the enzymatic reaction. This light can be detected using X-ray film or CCD imaging.

Advansta also offers a full selection of buffers, antibodies, blotting membranes, film and accessories optimized to work with the WesternBright HRP substrates. Conduct Western blots with confidence, drawing on Advansta's expertise to obtain the best data from your Wetsern blot experiments.

Learn more about Advansta's HRP substrates below, or use our substrate selection guide to determine which substrate is best suited to your experiment.

Substrates for every situation

WesternBright ECL provides a strong, long-lived signal for routine blots imaged using film. More sensitive than Amersham™ ECL™ or Pierce© ECL (Figure 2), WesternBright allows you to use less sample and antibody, saving precious resources and saving money.

Figure 2. WesternBright ECL detects equal amounts of protein with up to ten times less antibody. Duplicate slot blots containing serial dilutions of transferrin protein were probed with the antibody dilutions shown, and detected with either WesternBright ECL of Pierce ECL (Thermo Scientific) according to the manufacturers' instructions. The blots were imaged simultaneously on the same piece of film. WesternBright ECL produced the same signal with ten time less primary and five times less secondary antibody.

WesternBright Quantum is optimized for CCD imaging, and produces a signal that is linear with respect to protein amount over 3 orders of magnitude (Figure 3). WesternBright Quantum does not exhibit substrate depletion (inverse bands) at high protein amounts, and is extremely sensitive to detect attomoles of protein per band. For experiments aimed at quantifying differences in protein levels among samples, there is no better substrate available.

Figure 3. WesternBright Quantum has the largest usable linear dynamic range. Identical Western blots containing serial dilutions of transferrin were probed with a rabbit-anti-transferrin primary antibody, and a goat-anti-rabbit secondary antibody conjugated to HRP. The blots were incubated with chemiluminescent substrates as recommended by each manufacturer. All blots were simultaneously imaged for 2 minutes on a CCD imager. Band intensities were measured and plotted against protein concentration, and bands that fell on the linear part of the curve for each substrate are indicated with an orange line drawn over the bands in the figure.

WesternBright Sirius combines extreme sensitivity with quantitative signal, enabling detection of bands not visualized with other commercially available substrates.