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Choose chemiluminescent substrates for western blot detection based on sensitivity, protein abundance, and imaging requirements. Compare ECL substrates and chemiluminescent detection solutions to optimize signal intensity, sensitivity, and workflow compatibility across western blot applications. This page is for laboratories selecting chemiluminescent detection solutions for routine western blotting, low-abundance protein targets, or high-sensitivity applications.
Explore the western blot workflow
What is chemiluminescent detection?
Chemiluminescent detection is a high-sensitivity method used in western blotting to visualize proteins using enzyme-labeled antibodies and luminol-based chemiluminescent substrates. In western blot chemiluminescence detection workflows, horseradish peroxidase (HRP)- or alkaline phosphate (AP)-conjugated antibodies react with a chemiluminescent substrate to generate light that can be captured using imaging systems or X-ray film to visualize protein bands.
ECL (enhanced chemiluminescence) provides high sensitivity, strong signal amplification, and broad compatibility with HRP-based workflows. Depending on the chemiluminescent substrate selected, this approach can enable protein detection from attogram to picogram levels.
Use chemiluminescent detection when:
- Detecting low- or very low-abundance proteins
- Needing high sensitivity and signal amplification
- Performing quantitative or semi-quantitative analysis
- Working with limited or precious samples
Consider other methods if:
- You want simple visualization without imaging = Chromogenic
- You need multiplex detection = Fluorescent
Thermo Fisher Scientific offers several chemiluminescent substrates, each designed to support different experimental sensitivity and workflow needs.
Compare chemiluminescent substrates
SuperSignal chemiluminescent substrates support a range of western blot detection needs, from routine protein analysis to ultra-sensitive detection of low-abundance targets.
Choosing the right chemiluminescent substrate:
- For maximum sensitivity and very low abundance proteins: choose SuperSignal West Atto
- For high-sensitivity detection of low abundance proteins: choose SuperSignal West Femto
- For quantitative workflows or longer-lasting signal detection: choose SuperSignal West Dura
- For routine western blot workflows and general protein detection: choose SuperSignal West Pico PLUS
Use the comparison table below to select the right chemiluminescent substrate based on sensitivity, workflow fit, and recommended applications.
 SuperSignal West Pico PLUS |
 SuperSignal West Dura |
 SuperSignal West Femto |
 SuperSignal West Atto |
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| Recommended use | Routine western blot applications | Quantitative western blotting or extended signal detection | High-sensitivity detection of low-expression proteins | Maximum sensitivity for extremely low-abundant proteins |
| Sensitivity |
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| Signal characteristics | Reliable signal for general protein detection | Long-lasting signal for repeated imaging | Strong signal intensity with standard workflows | Maximum signal sensitivity |
Additional ECL substrates
| Recommended use | Sensitivity | |
|---|---|---|
| Pierce ECL | Routine detection of high-abundance protein targets when sample availability is not limiting | Low picogram |
| Pierce ECL Plus | Quantitative western blot workflows requiring chemiluminescent or chemifluorescent detection | Low picogram to mid-femtogram |
Additional products for HRP-based detection
| Description | |
|---|---|
| SuperSignal Western Blot Enhancer | Western blot signal enhancer and primary antibody diluent that can increase chemiluminescent signal intensity and sensitivity by 3- to 10-fold. Exceptional for improving detection of low-abundance proteins and weak signals in HRP-based western blot workflows. |
| Clean-Blot IP Detection Kit | Complete kit that helps reduce background from denatured IgG and improve signal-to-noise ratio in immunoprecipitation western blot workflows. |
| Clean-Blot IP Reagent | Detection reagent that helps reduce background from denatured IgG and improve target protein visualization in immunoprecipitation western blot workflows. |
Trial-size substrate kits
Compare multiple chemiluminescent substrates before committing to a larger format. Trial-size substrate kits allow researchers to compare chemiluminescent substrate sensitivity and signal characteristics before selecting a substrate for routine western blot workflows.
| SuperSignal Western Blot Substrate Bundle, Atto + trial-size Pico PLUS | SuperSignal Western Blot Substrate Bundle, Pico PLUS + trial-size Atto | SuperSignal Western Blot Substrate Bundle, Femto + trial-size Atto | SuperSignal Western Blot Substrate Trial Pack, Atto & Pico PLUS |
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Combo kit includes:
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Combo kit includes:
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Combo kit includes:
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Combo kit includes:
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Chemiluminescent substrate performance
Chemiluminescent detection relies on HRP- or AP-conjugated antibodies reacting with chemiluminescent substrates to generate light signals that are captured using imaging systems or film (Figure 1). Differences in substrate sensitivity, signal duration, and dynamic range can significantly affect western blot performance and should be considered when selecting a substrate.
Figure 1. Chemiluminescent western blot detection workflow. HRP- or AP-conjugated antibodies react with chemiluminescent substrates to generate light signals that are captured using imaging systems or film for protein visualization and analysis.
Chemiluminescent substrates are engineered for:
- Higher sensitivity—detect proteins from attogram to picogram levels
- Longer signal duration—support reliable imaging and reproducible results over extended exposure times
- Wide dynamic range—enable accurate detection and quantitation across a broad range of protein expression levels
- Consistent performance—deliver reproducible results across experiments
SuperSignal West Atto chemiluminescent substrate enables ultra-sensitive, attogram-level protein detection with strong signal-to-noise performance for western blot analysis. It allows clear detection across decreasing protein loads while maintaining sensitivity even at low concentrations of primary antibody dilutions. This combination makes it excellent for very low-abundance targets and workflows where both sample and antibody are limited.
Ultra-sensitive chemiluminescent detection for low-abundance proteins
SuperSignal West Atto enables detection of low-abundance proteins with high sensitivity and strong signal-to-noise, even at reduced lysate loads (0.04 µg), maintaining clear band visualization and reliable signal detection across a range of protein concentrations (Figure 2).
Figure 2. SuperSignal West Atto substrate allows use of very dilute samples. HeLa cell lysates were separated on Novex Tris-Glycine gels and transferred to nitrocellulose membranes using the Power Blotter transfer device with Power Blotter Select Transfer Stacks. The blots were incubated with mouse Anti p23, followed by goat anti-mouse HRP conjugate. Finally, blot was incubated with SuperSignal West Atto Substrate per product instructions, then acquired using the iBright FL Imaging System.
Maintain sensitivity with reduced antibody usage
SuperSignal West Atto chemiluminescent substrate maintains high sensitivity with significantly reduced primary antibody concentration, enabling reliable detection when antibody availability is limited or samples are difficult to obtain (Figure 3).
Figure 3. SuperSignal West Atto enables sensitive detection of Hsp90 in HeLa cell lysates while preserving valuable antibody reagents. Lysates (lanes 1–9) were loaded in decreasing amounts and transferred to nitrocellulose membranes using Power Blotter Select Transfer Stacks. Membranes were probed with anti-Hsp90 primary antibody at dilutions of 1:1,000 and 1:20,000, followed by an HRP-conjugated secondary antibody. Signals were developed using SuperSignal West Atto Substrate and imaged on the iBright FL Imaging System.
Workflow tip
Enhance detection of low-abundance proteins and dilute samples
When sample quantity is limited or protein expression is low, pairing WedgeWell format protein gels with SuperSignal West Atto substrate maximizes sensitivity and for improved detection of low-abundance proteins.
Explore WedgeWell protein gels
SuperSignal West Dura Extended Duration Substrate for HRP is optimized for high sensitivity, wide dynamic range, and long-lasting signal, making it excellent for quantitative western blot applications across a range of protein abundances. It enables reliable detection from medium- to low-expression targets with mid-femtogram sensitivity while maintaining strong linearity for accurate quantitation.
Quantitative chemiluminescent performance across protein abundance
SuperSignal West Dura demonstrates sensitive detection over a range of cellular protein abundance levels. Ras10 (low abundance), p23 (medium abundance), and HSP90 (high abundance) proteins were detected by western blot analysis using decreasing sample loads, illustrating strong signal detection even at reduced protein concentrations (Figure 4A). Quantitative analysis demonstrates excellent linearity (R² > 0.99) across multiple targets, supporting accurate detection and quantitation across a broad dynamic range (Figure 4B).
Figure 4. Obtain quantitative western blots with SuperSignal West Dura Substrate. (A) A 3-fold serial dilution of A431 or A549 lysate was loaded onto a Bolt 4-12% Bis-Tris-Plus gel and electrophoresed at 200V for 20 minutes. Gels were transferred to nitrocellulose membranes using the iBlot 2 Transfer Device. Blots were processed using the Bandmate Automated Western Blot Processor. Membranes were blocked in 1X Clear Milk Blocking Buffer. Each membrane was probed with a single primary antibody diluted in 1X Clear Milk Blocking Buffer with Mouse anti-p23 (1:25,000), rabbit anti-Hsp90 (1:25,000), and mouse anti-Pan Ras10 (1:5,000) followed by an incubation with secondary antibodies Stabilized Goat anti-Rabbit HRP 10µg/mL (1:500) or Stabilized Goat anti-Mouse HRP 10 µg/mL (1:500). SuperSignal West Dura substrate was used for detection. (B) Signal intensity correlated with lysate load for Ras10, p23, and HSP90 proteins.
Mid-femtogram detection
SuperSignal West Dura enables mid-femtogram sensitivity, supporting detection of low-expression proteins with clear, distinguishable signal (Figure 5). Long-lasting signal duration supports reliable image acquisition using film or digital imaging systems while helping minimize the need for repeated exposures. Even at low protein amounts, SuperSignal West Dura substrate maintains clear band visualization and consistent signal intensity for sensitive protein detection workflows.
Figure 5. Obtain mid-femtogram detection with SuperSignal West Dura substrate. Turbo GFP-His-HA-FLAG was diluted in electrophoresis reducing sample buffer. Lane 1 contained 1,000 fg of Turbo GFP. Six 1:1 serial dilutions were then prepared and applied at 10 μL/well. After electrophoresis, proteins were transferred to PVDF membrane, and then the membrane was blocked with SuperBlock T20 (TBS) Blocking Buffer. The membrane was incubated with biotinylated anti-His antibody at 1 μg/mL and then with Invitrogen Streptavidin, HRP Conjugate at 1:50,000 dilution. SuperSignal West Dura substrate was used for detection.
SuperSignal West Pico PLUS Substrate enables enhanced sensitivity and robust signal performance for routine western blot applications. It helps provide reliable detection across a wide range of protein expression levels and maintains consistent signal over time, making it well suited for everyday workflows or when optimizing new or unoptimized assays.
Low-picogram to mid-femtogram detection
SuperSignal West Pico PLUS enables reliable detection across a broad range of protein concentrations. Clear band detection is achieved from low-picogram to mid-femtogram protein levels, supporting sensitive detection in routine western blot experiments (Figure 6).
Figure 6. Low-picogram to mid-femtogram detection with SuperSignal West Pico PLUS substrate. Turbo GFP-His-HA-Flag was diluted in electrophoresis reducing sample buffer. Lane 1 contained 10 pg of purified protein with serial dilutions prepared 1:1 and applied at 10 µL/well. After electrophoresis, protein transfer to nitrocellulose membrane, and blocking, the membrane was incubated with anti-His antibody at 1 µg/mL, followed by incubation with Goat anti-Mouse HRP Conjugate at 100 ng/mL. SuperSignal West Pico PLUS substrate was used for detection.
Reliable detection across proteins with different abundance levels
SuperSignal West Pico PLUS helps deliver robust, sustained signal intensity across multiple exposure time points while enabling reliable detection of proteins spanning a wide range of protein abundance levels, from highly abundant target β-catenin, to moderately expressed protein STAT3, to low-abundance target WNT1. Additionally, this substrate maintains signal intensity across multiple time points, enabling consistent detection and flexible exposure times without rapid signal loss (Figure 7).
Figure 7. Robust signal with SuperSignal West Pico PLUS substrate. Detection of the indicated targets was performed using 2-fold serial dilutions of HEK293 lysates (β-catenin and STAT3; starting at 4 µg total protein/well) or HeLa lysates (WNT1; starting at 20 µg total protein/well). Following separation by SDS-PAGE, proteins were transferred with Invitrogen Power Blotter. The membranes were blocked with 5% nonfat dry milk dissolved in Pierce 20X TBS Tween 20 Buffer, and incubated with Invitrogen antibodies against beta-Catenin, STAT3, or WNT1 followed by incubation with Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP Conjugate at a concentration of 20 ng/mL. Chemiluminescent detection was performed following a 5-minute incubation with SuperSignal West Pico PLUS substrate. Signal was captured on film at the indicated time points after addition of substrate.
Fast western blotting kits
Pierce Fast Western Blot kits combine optimized blocking buffers, antibodies, and chemiluminescent substrates to reduce western blot workflow time from approximately 4 hours to about 55 minutes. These kits help minimize hands-on time while maintaining sensitivity and performance comparable to traditional western blotting workflows.
Fast Western Blot Kits are an excellent choice when:
- Faster western blot results are required
- Minimizing hands-on time is important
- Working with routine western blot workflows
- Standardized reagent performance is preferred
| Fast Western Blot Kit, SuperSignal West Pico | Fast Western Blot Kit, Pierce ECL | |
|---|---|---|
| Best suited for | Higher sensitivity detection |
Routine western blot detection |
| Sensitivity | Low picogram | Picogram |
Figure 8. Fast western versus tradition western blot workflow.
HRP vs alkaline phosphatase (AP) substrates
While chemiluminescent detection typically uses HRP, this method can also use alkaline phosphatase (AP). AP substrates are commonly used in specialized workflows where signal stability is more important than maximum sensitivity.
When should you use AP chemiluminescent substrates?
Use AP chemiluminescent substrates when:
- Longer-lasting signal is required for extended imaging
- Lower background is required in specific experimental conditions
- Slower signal development is preferred for workflow control
- Performing repeated image acquisition over longer periods
HRP substrates are generally preferred when maximum sensitivity and rapid signal generation are required.
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Description |
Ready-to-use AP chemiluminescent substrate for extended imaging, stable signal performance, and low-background applications |
Sensitivity |
Low picogram |
Duration |
Long-lasting signal (hours to days) |
Recommended membrane compatibility |
PVDF membranes Add Nitro Block II (sold separately) to substrate for use with nitrocellulose membranes |
Additional AP chemiluminescent detection kits
For AP-based chromogenic workflows, WesternBreeze chemiluminescent kits are available for mouse, rabbit, and goat primary antibodies.
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Description |
Complete AP chemiluminescent western blot detection kit for mouse primary antibodies, compatible with nitrocellulose or PVDF membranes. |
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Complete AP chemiluminescent western blot detection kit for rabbit primary antibodies compatible with nitrocellulose or PVDF membranes. |
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Complete AP chemiluminescent western blot detection kit for goat primary antibodies compatible with nitrocellulose or PVDF membranes. |
Specialized antibody formats for western blotting
Browse antibodies for chemiluminescent western blot detection.
Ordering information
Complete the western blot workflow
Explore additional solutions that support each step of the western blot workflow from protein detection to data analysis:
Frequently asked questions
ECL is a chemiluminescent detection method using HRP and luminol substrates to generate light for protein visualization.
Choose based on protein abundance, sensitivity needs, and required signal duration:
- Use SuperSignal West Atto for very low-abundance proteins when maximum sensitivity is required.
- Use SuperSignal West Femto for low-abundance proteins when high sensitivity is needed.
- Use SuperSignal West Dura when you need extended signal stability or repeat imaging.
- Use SuperSignal West Pico PLUS for routine western blot workflows and general protein detection.
SuperSignal West Atto offers the highest sensitivity, detecting proteins at attogram levels.
Yes. Chemiluminescent detection can be used for quantitative western blot analysis when signals are captured within the linear range of the assay. SuperSignal West Dura provides strong linearity, broad dynamic range, and reproducible signal performance for accurate quantitation across a range of protein expression levels.
HRP substrates provide higher sensitivity and faster signal generation, while AP substrates offer longer-lasting signal and are used in specialized applications.
Western blot educational resources
Access resources to help optimize western blot detection strategies, troubleshoot experiments, and improve data quality.
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