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Introducing a Western blotting substrate that’s chemiluminescent and chemifluorescent
Detect your target by film, CCD cameras or laser-based imagers.
The Thermo Scientific Pierce ECL Plus Substrate is a chemiluminescent and chemifluorescent peroxidase substrate for Western blotting. The Pierce ECL Plus Substrate generates acridinium esters when it reacts with horseradish peroxidase (HRP), producing a strong signal that is equal to or better than similar commercially available substrates (Figure 1). The ester intermediates react with peroxide and produce a strong and sustained chemiluminescent signal that can be captured by X-ray film or a CCD camera. A robust fluorescent signal is also produced, which can be captured with fluorescence imagers (Figure 2).
Figure 1. Thermo Scientific Pierce ECL Plus Substrate performs as well or better than substrates from other suppliers. HeLa cell lysate was diluted in SDS-PAGE sample buffer and heated to 95˚C for 5 minutes. Lane 1 contained 10μg of total protein. Four 1:1 dilutions were prepared and applied to Lanes 2-5 at 10μL/well. The proteins were transferred to nitrocellulose membranes (Part No. 88013). The membranes were blocked with 5% milk in Tris-buffered saline with Tween*-20 Detergent. Rabbit anti-hsp86 antibody (Ab No. PA3-013) diluted 1:1000 and goat anti-rabbit HRP (Part No. 31460) at 6.6ng/mL were used for target detection. Signal was detected using the indicated substrates and Thermo Scientific CL-XPosure Film (Part No. 34090).
Figure 2. One substrate detected by three methods. Blots were prepared as described in Figure 1. The target was detected with rabbit anti-MAP kinase (EMD Millipore) diluted 1:1000, goat anti-rabbit HRP (Part No. 31460) at 6.6ng/mL and Pierce ECL Plus Substrate. The membranes were exposed to CL-XPosure Film for five seconds and scanned using the Typhoon* 9410 Variable Mode Imager (excitation at 457nm, emission at 510nm) and Syngene* G:Box iChemiXT Imager (1 minute exposure).
Pierce ECL Plus Substrate enables detection of low-picogram amounts of target protein (Figure 3) on nitrocellulose and PVDF membranes when probed with the appropriate primary and secondary antibody concentrations. This versatile and reliable Western blotting substrate has a broad dynamic range and excellent sensitivity combined with a long-lasting signal (Figure 4).
Figure 3. Chemiluminescent signal can be detected for hours after substrate incubation. Blots were prepared as described in Figure 1. The target was detected with rabbit anti-hsp86 (Ab No. PA3-013) diluted 1:1000 and goat anti-rabbit HRP (Part No. 31460) at 6.6ng/mL. Signal was detected with CL-XPosure Film exposed at the indicated times.
Figure 4. Detect low-picogram amounts of target protein. GST (500ng) was loaded onto Lane 1 and serially diluted 1:1 and loaded onto Lanes 2-9 (10μL/well). Proteins were transferred to nitrocellulose membrane (Part No. 88013) and Thermo Scientific SuperBlock Blocking Buffer TBS (Part No. 37535) was used to block nonspecific binding sites. GST was detected with biotinylated anti-GST (Santa Cruz) at 0.5μg/mL and Thermo Scientific Pierce High Sensitivity Streptavidin-HRP (Part No. 21130) diluted 1:60,000. Signal was detected with CL-XPosure Film exposed for 5 seconds.