Pierce™ ECL Plus Western Blotting Substrate

Thermo Scientific Pierce ECL Plus Substrate is an acridan-based chemiluminescent and chemifluorescent HRP substrate for Western blot detection using X-ray film or CCD- or laser-based imagers.
Features of ECL Plus Substrate:

• Easy to use—can be substituted for the discontinued GE ECL Plus Substrate without any re-optimization
• Higher sensitivity—detect targets down to the low-picogram level
• Longer signal duration—sustained light output for as long as 5 hours
• More imaging options—X-ray, CCD or laser-based imagers
• More affordable—high quality and performance at a lower price than other suppliers' similar substrates

Pierce ECL Plus Substrate generates acridinium esters when it reacts with HRP. As these ester intermediates react with peroxide, they produce a strong and sustained chemiluminescent signal that can be captured by X-ray film CCD imaging. The substrate also produces a robust fluorescent signal that can be captured with fluorescence imagers.

Pierce ECL Plus Substrate enables the detection of low-picogram amounts of target protein on nitrocellulose or PVDF membrane when probed with appropriate primary and secondary antibody concentrations. Its broad dynamic range, high sensitivity and long-lasting signal make Pierce ECL Plus Substrate an excellent choice for Western blot analysis



 

Obtain excellent detection of target proteins with ECL Plus Substrate
Lysates from the cell lines indicated below were diluted in electrophoresis sample buffer and heated to 95°C for 5 minutes. Lane 1 contained 10μg of total protein, and seven 1:1 serial dilutions were loaded at 10μL/well. After electrophoresis, proteins were transferred to nitrocellulose membrane (Part No. 88013), and then the membranes were blocked with 5% milk in TBST. The membranes were incubated with primary antibody at 1:1000 dilution and then with the secondary antibody at 6.6ng/mL (1:150,000 dilution of 1mg/mL stock solution). A. Target: β-Catenin in HeLa cells; Primary Antibody: Rabbit Anti-β-Catenin (Thermo Scientific Lab Vision Part No. RB-1491-PABX); Secondary: HRP-conjugated Goat Anti-Rabbit IgG (Part No. 31460); B. Target: ERK1 in K562 cells; Primary Antibody: Mouse Anti-ERK1 (Part No. MA113041); Secondary Antibody: HRP-conjugated Goat Anti-Mouse IgG (Part No. 31430); C. Target: PP2A in A549 cells; Primary Antibody: Mouse Anti-PP2A (Millipore); Secondary Antibody: HRP-conjugated Goat Anti-Mouse IgG (Part No. 31430). CL-XPosure Film (Part No. 34090) and Pierce ECL Plus Substrate were used for detection. Exposure time was 5 minutes.



 

Obtain picogram detection and broad dynamic range using ECL Plus Substrate
GST (Part No. 20237) was diluted in electrophoresis sample buffer. Lane 1 contained 500ng of GST. Nine 1:1 serial dilutions were then prepared and applied at 10μL/well. After electrophoresis, proteins were transferred to nitrocellulose membrane (Part No. 88013), and then the membrane was blocked with Thermo Scientific SuperBlock in TBS Buffer (Part No. 37535). The membrane was incubated with Biotinylated Anti-GST (Santa Cruz) at 0.5μg/mL and then with Thermo Scientific Pierce High Sensitivity Streptavidin-HRP (Part No. 21130) at 1:60,000 dilution. Pierce ECL Plus Substrate (Part No. 32132) was used for detection.

ECL Plus Substrate delivers high performance and a long-lasting signal using three methods of detection
Hsp86 was detected in HeLa cell lysates according to the following protocol. The lysates were diluted in electrophoresis sample buffer and heated to 95°C for 5 minutes. For each blot, lane 1 contained 10μg of total protein, and four 1:1 serial dilutions were applied to lanes 2-5 at 10μL/well. After electrophoresis, proteins were transferred to nitrocellulose membranes (Part No. 88013), and then the membranes were blocked with 5% milk in TBST. The membranes were incubated with Rabbit Anti-Hsp86 (Part No. PA3-013) at 1:1000 dilution and then with HRP-conjugated Goat Anti-Rabbit IgG (Part No. 31460) at 6.6ng/mL (1:150,000 dilution of 1mg/mL stock solution). (A) Blots were prepared using Pierce ECL Plus Substrate (Part No. 32132) or substrates from other suppliers, and then all blots were exposed to Thermo Scientific CL-XPosure Film (Part No. 34090). (B) Blots were prepared using Pierce ECL Plus Substrate and exposed to CL-XPosure Film at the indicated time points. (C) The membranes were prepared using Pierce ECL Plus Substrate and exposed to CL-XPosure Film for five seconds and scanned using the Syngene™ G:Box iChemiXT Imager (CCD; 1 minute exposure) and the Typhoon™ 9410 Variable Mode Imager (Laser-based imager; Excitation at 457nm, Emission at 510nm).

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