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Western Blot Chemiluminescent Substrate Selection Guide

Choose an appropriate ECL reagent for Western blot detection.

ECL substrate selection guideChemiluminescence is the Western blot detection method of choice in most protein laboratories, as it provides the greatest sensitivity and convenience for detection with film or digital imaging equipment. Several varieties of luminol-based chemiluminescent substrates are available, and choosing among them for a particular experiment or available equipment can seem overwhelming.

Chemiluminescent substrates for horseradish peroxidase (HRP) are two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. To make a working solution, the equal volumes of the two components are mixed together. When incubated with a blot on which HRP-conjugated antibodies (or other probes) are bound, a chemical reaction produces light that can be detected by film or sensitive camera.

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Protein Methods Library Home


Overview of Western Blotting

Chemiluminescent Western Blots

Related literature...
Western Blotting Handbook
Western Blot Handbook

Criteria for Choosing Chemiluminescent Substrates

Chemiluminescent substrates differ in price, ease-of-use, sensitivity (i.e., lower limit of detection) and compatibility with imaging equipment. More sensitive substrates cost more, so it might be natural to assume that one should use the most sensitive substrate that can be afforded. However, this is not necessarily correct because ease-of-use is an equally important consideration.

Although the actual procedure for preparing substrate working solution and applying it to a blot is practically identical for all chemiluminescent substrates, each substrate has unique requirements for optimization. In general, it is more difficult to optimize Western blot protocols with more sensitive substrates because even the tiniest flaws in blocking or non-specific binding are detectable. No researcher wants to spend more time than absolutely necessary optimizing antibody dilutions and exposure times to obtain a useful result.

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Western Blot Substrates

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Tech Tip #32: Guide to enzyme substrates for Western blotting

Available Chemiluminescent Substrate Reagents

Thermo Scientific Pierce ECL and SuperSignal Substrates

We offer five types of chemiluminescent substrates for Western blot detection with horseradish peroxidase enzyme (HRP):

  • Pierce ECL Substrate
    • A less expensive direct alternative to the once-popular GE Healthcare ECL (commonly called "Amersham ECL")
    • Appropriate for applications in which very abundant proteins (i.e., actin, tubulin, etc) are being probed or for experiments that have already been optimized with the GE Healthcare product
  • Pierce ECL Plus Substrate
    • Less-expensive direct alternative to GE Healthcare ECL Plus Substrate
    • Offers greater sensitivity and requires less antibody than ECL substrates
  • SuperSignal West Pico Chemiluminescent Substrate
    • Provides excellent performance for the range of target protein amounts typical of routine experiments
    • Easily optimized to detect with greater senstivity than entry-level ECL substrates
  • SuperSignal West Dura Extended Duration Substrate
    • Offers high sensitivity and long-duration signal that facilitate CCD-camera and other digital imaging analysis
    • 24-hour signal output allows researchers to make multiple exposures with imaging equipment
  • SuperSignal West Femto Maximum Sensitivity Substrate
    • One of the most sensitive substrates available for Western blotting of any kind. When properly optimized, the lower detection limit rivals radioactive detection methods using 32P
    • However, without proper optimization, it easy to overwhelm the system with protein and enzyme, resulting in high background and false negative results

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myECL Imager for ECL blot documentation
myECL Imager


All Western Blot Substrates

Lumi-Phos WB Substrate
A chemiluminescent substrate
for alkaline phosphatase (AP)

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Tech Tip #32: Guide to enzyme substrates for Western blotting

Chemiluminescent HRP Substrates Comparison Chart. Sensitivity and antibody dilutions have been generalized as a means to begin optimization. Individual assays may require conditions outside the ranges suggested here.
Thermo Scientific Substrate Primary Benefits Sensitivity Sensitivity
Rating
Signal Duration (hours) Recommended
Antibody Dilution
Pierce ECL Same signal and lower price than other ECL substrates Low picogram 1 1 to 2 1°: 0.2 to 10μg/mL
2°: 0.07 to 1μg/mL
SuperSignal West Pico Better signal and lower price than competing ECL substrates Low picogram 2 6 to 8 1°: 0.2 to 1μg/mL
2°: 10 to 50ng/mL
Pierce ECL Plus Same signal and lower price than competing ECL Plus substrates Low picogram to mid-femtogram 3 5 1°: 0.05 to 1μg/mL
2°: 5 to 40ng/mL
SuperSignal West Dura Best for use with imaging equipment Mid-femtogram 4 24 1°: 0.02 to 1μg/mL
2°: 4 to 20ng/mL
SuperSignal West Femto Most sensitive substrate for HRP detection Low to mid-femtogram 5 6 to 8 1°: 10 to 200ng/mL
2°: 2 to 10ng/mL
† Sensitivity Rating: 1=lowest, 5=highest

 

Related Chemiluminescent Reagents and Kits

In addition to the reagent products listed above, complete Western blotting kits are available for particular applications using Pierce ECL and SuperSignal Substrates. These kits include the chemiluminescent substrate plus validated components such as blocking buffer and secondary antibodies.

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Pierce Fast Western Blot Kits

SuperSignal Western Blotting Kits

Also available are kits for direct in-gel detection of HRP-conjugated probes by chemiluminescence. Although not as sensitive as a typical Western blot, the in-gel system allows results to be obtained in times similar to the Fast Western (Thermo Scientific), SNAP i.d. (Millipore), and iBlot (Invitrogen) detection systems when used with compatible polyacrylamide gels. There is also the added advantage of reduced waste from buffers and consumables, such as filter paper and membranes.

Other specialized kits include those specific for glycosylation, His-tagged proteins far-Western assays.

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In-Gel Detection Kits

Far-Western Blotting Kit

SuperSignal HisProbe Kit

O-GlcNAc Western Blot Kit

Certain primary antibodies and reagents have been optimized for use with SuperSignal West Dura Extended Duration Substrate to perform and validate siRNA-knockdown by Western blot.

Specific formulations and kits of SuperSignal Chemiluminescent Substrates are available for nucleic acid techniques, including EMSA and Southern blotting. These products depend upon the use of biotin-labeled DNA or RNA targets and hybridization probes, which are then detected via HRP-conjugated streptavidin.

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Tools for siRNA-knockdown confirmation by Western blotting

Nucleic Acid Labeling and Detection Kits

Compatible Instrumentation

Signal from ECL substrates can be captured and documented using X-ray film or a capable CCD camera imager.

myECL Imager for ECL blot documentation

The Thermo Scientific myECL Imager is a stylish, powerful and easy-to-use blot and gel documentation instrument for sensitive, multimode image capture and analysis via an intuitive touchscreen interface and advanced integrated software.


Other instruments capable of ECL signal documentation. The following instruments listed here are capable of documenting signal from luminol-based substrates, such as the Thermo Scientific SuperSignal Substrates.
Supplier Instrument
Alpha Innotech FluorChem and ChemiImager
Bio-Rad ChemiDocXRS and VersaDoc Imaging
Systems, Model 4000 and 5000
UVP Chemi, BioChemi and OptiChemi Systems
Kodak Image Station 2000MM
Fotodyne FOTO/Analyst Luminary
Fujifilm LAS-3000 Imaging System
UVItec Ltd. UVIchemi and UVIprochemi
Syngene G:BOX and GeneGnome Models

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myECL Imager

myImageAnalysis Software

CL-Xposure X-ray Film

Browse all Western blot reagents, kits and equipment


Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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