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Pierce ECL Plus Western Blotting Substrate


Recommended HRP substrate for fluorescence imagers.

Pierce ECL Plus SubstrateThermo 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.

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.

This substrate is also sold through VWR (US, Canada and Scandinavia only) and Fisher Scientific as Pierce ECL 2 Substrate (Part No. PI80196) – the same formulation as Pierce ECL Plus Substrate but specially packaged for these vendors.

Highlights:

  • 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

Product Details:

Thermo Scientific Pierce ECL Plus Substrate delivers high performance and provides a long-lasting signal using three methods of detection
Thermo Scientific Pierce 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).

Obtain picogram detection and broad dynamic range using Thermo Scientific Pierce ECL Plus
Obtain picogram detection and broad dynamic range using Thermo Scientific Pierce 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.

Obtain excellent detection of target proteins with Thermo Scientific Pierce ECL Plus Substrate
Obtain excellent detection of target proteins with Thermo Scientific Pierce 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.

Related Resources:

Western blotting and detection technical guide
Western blot video tutorials

Related Products:

Selection Guide for Chemiluminescent Western Blot Substrates
Blotting Equipment, Membranes and Signal Enhancers
CL-XPosure Film (X-ray film for chemiluminescence)


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
32132 Pierce ECL Plus Western Blotting Substrate
Formulation: Proprietary acridan and peroxide solutions
Sufficient For: 10 mini-gel blots; 1000cm2 of membrane

Kit Contents:
Detection Reagent A, 100mL
Detection Reagent B, 2.5mL
100mL kit Product Instructions for product #32132 Pierce ECL Plus Western Blotting Substrate MSDS for product #32132 Pierce ECL Plus Western Blotting Substrate Certificate of Analysis for product #32132 Pierce ECL Plus Western Blotting Substrate $261.00

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32132X3 Pierce ECL Plus Western Blotting Substrate
Formulation: Proprietary acridan and peroxide solutions
Sufficient For: 30 mini-gel blots; 3000cm2 of membrane

Kit Contents:
Detection Reagent A, 3 x 100mL
Detection Reagent B, 3 x 2.5mL
300mL kit MSDS for product #32132X3 Pierce ECL Plus Western Blotting Substrate $522.00

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32134 Pierce ECL Plus Western Blotting Substrate
Formulation: Proprietary acridan and peroxide solutions
Sufficient For: 250cm2 of membrane

Kit Contents:
Detection Reagent A, 25mL
Detection Reagent B, 0.625mL
25mL kit Product Instructions for product #32134 Pierce ECL Plus Western Blotting Substrate MSDS for product #32134 Pierce ECL Plus Western Blotting Substrate Certificate of Analysis for product #32134 Pierce ECL Plus Western Blotting Substrate $86.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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