SuperSignal Chemiluminescent Substrates

ECL substrates for high-sensitivity western blot detection

Thermo Scientific SuperSignal ECL substrates are HRP substrates that offer excellent performance in western blotting applications, with longer light emission and stronger signal intensity than other luminol-based or acridan-based detection systems. Several varieties of ECL western blot kits are available, each designed to meet particular experiment needs. Learn more about the available ECL kits with the application data highlighted below.

Find the right chemiluminescent substrate

Western blot antibody dilution calculator

ECL substrates for western blotting

SuperSignal West AttoSuperSignal West DuraSuperSignal West Pico PlusPierce ECL 
SuperSignal West AttoSuperSignal West DuraSuperSignal West
Pico PLUS
Pierce ECL
Choose when
Detecting very low abundant protein targets, limited antibodies, or limited sample volumesPerforming quantitative western blots or long duration is neededFor routine western blots or new systems not yet optimizedDetecting high abundant protein targets and sample is abundant
Features
Highest sensitivity with less optimization requiredGood sensitivity with long duration and linearity for sensitive quantitationBetter signal and lower price than competing ECL substratesSame signal and lower price than other standard ECL substrates
Sensitivity
Low femtogram to high attogramMid-femtogramLow picogram to high femtogramLow picogram
Working solution stability
48 hr24 hr8 hr1 hr
Signal duration
6 hr24 hrUp to 24 hr1-2 hr
Recommended antibody dilutions
1°: 0.2–1 μg/mL
(1:1,000-1:5,000)
2°: 4–10 ng/mL
(1:100,000- 1:250,000)
1°: 0.02–1 μg/mL
(1:1,000-1:50,000)
2°: 4–20 ng/mL
(1:50,000- 1:250,000)
1°: 0.2–1 μg/mL
(1:1,000- 1:5,000)
2°: 10–50 ng/mL
(1:20,000-1:100,000)
1°: 0.2–10 μg/mL
(1:100-1:5,000)
2°: 0.07–1 μg/mL
(1:1,000-1:15,000)


Western blot ECL substrates

Enhanced chemiluminescence (ECL) substrates, are HRP substrates that are typically two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. To make a working solution, the two components are mixed together. When incubated with a blot on which HRP-conjugated antibodies (or other probes) are bound, a chemical reaction emits light at 425 nm which can be captured with x-ray film, CCD camera imaging devices and phosphorimagers that detect chemiluminescence. Light emission occurs only during the enzyme-substrate reaction; therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases.

We offer six types of ECL substrates for western blot detection with horseradish peroxidase enzyme (HRP). Each containing different enhancers to increase signal, linearity and duration compared to other western blot detection systems. Use the table above to select the most appropriate ECL kit based on abundance of your target protein of interest, abundance of sample containing the target protein, and the level of sensitivity and type of instrumentation available for detection.

Don’t know which ECL western blot kit is right for you? Use the chemiluminescent HRP substrate selection guide to choose the right HRP substrate for your research needs.

Why do our ECL kits have recommended antibody dilutions?

Under optimal western blotting conditions, a chemiluminescent signal can last for 6–24 hr. The level and duration of light generation depends on the specific substrate being used and the enzyme-to-substrate ratio in the system. Although the amount of substrate on a blot is relatively constant, the amount of enzyme present depends on the amount of antibody-conjugate added. Too much enzyme conjugate applied to a western blot system is one of the greatest causes of signal variability, dark background, short signal duration, and low sensitivity. A signal emission curve that decays slowly is desirable as it demonstrates that each component of the system has been optimized and allows reproducible results. A signal that decays too quickly can cause variability, low sensitivity, high background, and problems with signal documentation. A long-lasting signal minimizes variability in results due to transfer efficiency, different manufacturer lots of substrate, and other factors. The oxidation reaction of the HRP molecules with the luminol in the substrate produces free radicals in addition to the light being produced. An abundance of HRP in the system will create an abundance of free radicals, speeding the probability of HRP inactivation. Free radicals may also damage the antigen, antibodies, and the membrane, resulting in reduced effectiveness of re-probing.

SuperSignal West Pico Plus

SuperSignal West Pico PLUS provides excellent performance, versatility, and economy for routine western blotting needs. It is designed to work for the majority of western blots and recommended for detecting a new protein of interest when western blotting conditions are not yet optimized. SuperSignal West Pico PLUS Chemiluminescent Substrate offers better sensitivity, longer signal duration, and brighter intensity than our original SuperSignal West Pico Substrate and entry level ECL substrates.

SuperSignal West Pico PLUS substrate outperforms Bio-Rad™ Clarity™ substrate.

SuperSignal™ West Pico PLUS Chemiluminescent Substrate

Chemiluminescent substrate product comparison with SuperSignal West Pico PLUS substrate. Detection of the indicated targets was performed using 2-fold serial dilutions of HEK293 or HeLa cell lysates, starting with the amount indicated in parentheses. Chemiluminescent detection and substrate comparison was performed following a 5-minute incubation with either SuperSignal West Pico PLUS substrate or Bio-Rad Clarity substrates. See product page for additional details.

Low-picogram to high-femtogram detection

Low picogram to high femtogram detection

Low-picogram to high-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 the 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 (Cat. No. MA1-21315) at 1 µg/mL, followed by incubation with Goat anti-Mouse HRP Conjugate (Cat. No. 32430) at 100 ng/mL. SuperSignal West Pico PLUS substrate (Cat. No. 34577) was used for detection.

Robust signal

Robust signal

Robust signal with SuperSignal West Pico PLUS substrate. Detection of the indicated targets was performed using 2-fold serial dilutions of HEK293 or HeLa cell lysates, starting at 4 µg/well or 20 µg/well, respectively. Following separation by SDS-PAGE, proteins were transferred with Invitrogen Power Blotter (Cat. No. PB0013). The membranes were blocked with 5% nonfat dry milk dissolved in Pierce 20X TBS Tween 20 Buffer (Cat. No. 28360), and incubated with Invitrogen antibodies against beta-Catenin (Cat. No. MA1-300), STAT3 (Cat. No. MA1-13042), or WNT1 (Cat. No. MA5-15544) followed by incubation with Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP Conjugate (Cat. No. 31430) 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.

SuperSignal West Dura

Thermo Scientific SuperSignal West Dura Extended Duration Substrate for HRP is optimized for high sensitivity, wide dynamic range and long signal duration, making it ideal for sensitive quantitative western blots. Unlike substrates with signals that decline to barely detectable levels in 30–60 minutes, the signal produced with SuperSignal West Dura chemiluminescent substrate is stable for 24 hours.

Quantitative western blots for a range of target protein abundance

Quantitative western blot analysis for low, medium and high abundant proteins using SuperSignal West Dura ECL kit

Obtain quantitative western blots with SuperSignal West Dura Substrate. A 3-fold serial dilution of A431 or A549 lysate was loaded onto a Bolt 4-12% Bis-Tris-Plus gel (NW04122BOX) and electrophoresed at 200V for 20 minutes. Gels were transferred to nitrocellulose membranes using the iBlot 2 Transfer Device (P0, 7 min). Blots where processed using the Bandmate Automated Western Blot Processor. Membranes were blocked in 1X Clear Milk Blocking Buffer (Cat. No. 37587). Each membrane was probed with a single primary antibody diluted in 1X Clear milk. Mouse anti-p23 (1:25,000) (Cat. No. MA3-414), rabbit anti-Hsp90 (1:25,000) (Cat. No. PA3-013), mouse anti-Pan Ras10 (1:5,000) (Cat. No. MA1-012) and mouse anti-Mu-Calpain (1:5,000) (Cat. No. MA3-940), followed by an incubation with secondary antibodies Stabilized Goat anti-Rabbit HRP 10µg/mL (1:500) (Cat. No. 32460) or Stabilized Goat anti-Mouse HRP 10 µg/mL (1:5 00) (Cat. No. 32430). SuperSignal West Dura substrate (Cat. No. 34076) was used for detection.

Mid-femtogram detection

Obtain mid-femtogram detection with SuperSignal West Dura substrate

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. Seven 1:1 serial dilutions were then prepared and applied at 10 μL/well. After electrophoresis, proteins were transferred to PVDF membrane (Cat. No. 88518), and then the membrane was blocked with SuperBlock T20 (TBS) Blocking Buffer (Cat. No. 37536). The membrane was incubated with biotinylated anti-His antibody (Cat. No. MA1-21315-BTIN) at 1 μg/mL and then with Invitrogen Streptavidin, HRP Conjugate (Cat. No. 21126) at 1:50,000 dilution. SuperSignal West Dura substrate (Cat. No. 34076) was used for detection.

Better sensitivity and signal duration

Better sensitivity and signal duration with SuperSignal West Dura substrate

Better sensitivity and signal duration with SuperSignal West Dura substrate. A431 cell lysate was diluted in electrophoresis reducing sample buffer. Lane 1 contained 5 µg of A431 lysate. Five 1:1 serial dilutions were then prepared and applied at 10 μL/well. After electrophoresis, proteins were transferred to a nitrocellulose membrane using the Invitrogen Power Blotter (Cat. No. PB0013). Membranes were blocked with 5% milk in TBST Buffer. The membranes were incubated with Anti-beta-Catenin (Cat. No. MA1-300) at 0.3 μg/mL and then with Goat anti-Mouse HRP Conjugate (Cat. No. 32430) at 20 ng/mL. Identical blots were incubated in either SuperSignal West Dura substrate (Cat. No. 34076), Clarity™ Western ECL Blotting Substrate (Bio-Rad Laboratories, Inc.) or Immobilon™ Western Chemiluminescent HRP Substrate (EMD Millipore Corp.) according to respective manufacturer's instructions. 30-second exposures of the resulting blots were acquired.

SuperSignal West Atto

SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate is an ultrasensitive enhanced chemiluminescent (ECL) substrate that enables high attogram level protein detection by western blot analysis with the horseradish peroxidase (HRP) enzyme. It provides the highest-level sensitivity and better signal-to-noise ratios than other commercially available high-performance HRP substrates. It is the ideal choice for detection of very low-abundance targets or when using precious samples that require maximum levels of sensitivity.

Detect lower levels of protein

Figure 1. Chemiluminescent substrate product comparison with SuperSignal West Atto substrate.

  1. Akt (pan) detection in HeLa lysate was performed using HRP chemiluminescent substrates, SuperSignal West Atto, Bio-Rad Clarity Max, Amersham ECL Select, Millipore Immobiolon Western, Bio-Rad Clarity and standard ECL. The blots were developed using AKT (pan) monoclonal antibody (Cat. No. MA5-14916), followed by HRP-conjugated goat anti-rabbit IgG secondary antibody (Cat. No. 32460). Blots were incubated with respective substrates per product instructions. Images were captured using the iBright Imaging System.
  2. The signal-to-noise ratios for the western blot bands in the boxed area for SuperSinal West Atto, Bio-Rad Clarity Max, Amersham ECL Select, Millipore Immobilon Western, Bio-Rad Clarity and ECL are shown in bar graph form. SuperSignal West Atto provided the best signal-to-noise ratios across all protein loads. ECL didn’t have detectable signal above background noise after 0.625ug. Clarity and Immobilon didn’t have detectable signal above background noise after 0.313ug.

Use less samples

SuperSignal West Atto substrate allows use of very dilute samples. p23 in whole cell HeLa cell lysates was detected with SuperSignal West Atto, Bio-Rad Clarity or Millipore Immobion. Only 1.25 µg of cell lysate was needed to get strong signal with SuperSignal West Atto, whereas 20 µg of cell lysate was required to get the same level of detection with the other two substrates.

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 (Cat. No. PB3210). The blots were incubated with mouse Anti p23 (Cat. No. MA3-414), follow by goat anti-mouse HRP conjugate (Cat. No. 32430). Finally, respective blots were incubated with SuperSignal West Atto Substrate (Part No. A38558), Clarity™ Western ECL Blotting Substrate (Bio-Rad Laboratories, Inc.) or Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore Corp.) per product instructions. All blots were simultaneously acquired using the iBright FL Imaging System.

ECL substrates for western blotting

SuperSignal West AttoSuperSignal West DuraSuperSignal West Pico PlusPierce ECL 
SuperSignal West AttoSuperSignal West DuraSuperSignal West
Pico PLUS
Pierce ECL
Choose when
Detecting very low abundant protein targets, limited antibodies, or limited sample volumesPerforming quantitative western blots or long duration is neededFor routine western blots or new systems not yet optimizedDetecting high abundant protein targets and sample is abundant
Features
Highest sensitivity with less optimization requiredGood sensitivity with long duration and linearity for sensitive quantitationBetter signal and lower price than competing ECL substratesSame signal and lower price than other standard ECL substrates
Sensitivity
Low femtogram to high attogramMid-femtogramLow picogram to high femtogramLow picogram
Working solution stability
48 hr24 hr8 hr1 hr
Signal duration
6 hr24 hrUp to 24 hr1-2 hr
Recommended antibody dilutions
1°: 0.2–1 μg/mL
(1:1,000-1:5,000)
2°: 4–10 ng/mL
(1:100,000- 1:250,000)
1°: 0.02–1 μg/mL
(1:1,000-1:50,000)
2°: 4–20 ng/mL
(1:50,000- 1:250,000)
1°: 0.2–1 μg/mL
(1:1,000- 1:5,000)
2°: 10–50 ng/mL
(1:20,000-1:100,000)
1°: 0.2–10 μg/mL
(1:100-1:5,000)
2°: 0.07–1 μg/mL
(1:1,000-1:15,000)


Western blot ECL substrates

Enhanced chemiluminescence (ECL) substrates, are HRP substrates that are typically two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. To make a working solution, the two components are mixed together. When incubated with a blot on which HRP-conjugated antibodies (or other probes) are bound, a chemical reaction emits light at 425 nm which can be captured with x-ray film, CCD camera imaging devices and phosphorimagers that detect chemiluminescence. Light emission occurs only during the enzyme-substrate reaction; therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases.

We offer six types of ECL substrates for western blot detection with horseradish peroxidase enzyme (HRP). Each containing different enhancers to increase signal, linearity and duration compared to other western blot detection systems. Use the table above to select the most appropriate ECL kit based on abundance of your target protein of interest, abundance of sample containing the target protein, and the level of sensitivity and type of instrumentation available for detection.

Don’t know which ECL western blot kit is right for you? Use the chemiluminescent HRP substrate selection guide to choose the right HRP substrate for your research needs.

Why do our ECL kits have recommended antibody dilutions?

Under optimal western blotting conditions, a chemiluminescent signal can last for 6–24 hr. The level and duration of light generation depends on the specific substrate being used and the enzyme-to-substrate ratio in the system. Although the amount of substrate on a blot is relatively constant, the amount of enzyme present depends on the amount of antibody-conjugate added. Too much enzyme conjugate applied to a western blot system is one of the greatest causes of signal variability, dark background, short signal duration, and low sensitivity. A signal emission curve that decays slowly is desirable as it demonstrates that each component of the system has been optimized and allows reproducible results. A signal that decays too quickly can cause variability, low sensitivity, high background, and problems with signal documentation. A long-lasting signal minimizes variability in results due to transfer efficiency, different manufacturer lots of substrate, and other factors. The oxidation reaction of the HRP molecules with the luminol in the substrate produces free radicals in addition to the light being produced. An abundance of HRP in the system will create an abundance of free radicals, speeding the probability of HRP inactivation. Free radicals may also damage the antigen, antibodies, and the membrane, resulting in reduced effectiveness of re-probing.

SuperSignal West Pico Plus

SuperSignal West Pico PLUS provides excellent performance, versatility, and economy for routine western blotting needs. It is designed to work for the majority of western blots and recommended for detecting a new protein of interest when western blotting conditions are not yet optimized. SuperSignal West Pico PLUS Chemiluminescent Substrate offers better sensitivity, longer signal duration, and brighter intensity than our original SuperSignal West Pico Substrate and entry level ECL substrates.

SuperSignal West Pico PLUS substrate outperforms Bio-Rad™ Clarity™ substrate.

SuperSignal™ West Pico PLUS Chemiluminescent Substrate

Chemiluminescent substrate product comparison with SuperSignal West Pico PLUS substrate. Detection of the indicated targets was performed using 2-fold serial dilutions of HEK293 or HeLa cell lysates, starting with the amount indicated in parentheses. Chemiluminescent detection and substrate comparison was performed following a 5-minute incubation with either SuperSignal West Pico PLUS substrate or Bio-Rad Clarity substrates. See product page for additional details.

Low-picogram to high-femtogram detection

Low picogram to high femtogram detection

Low-picogram to high-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 the 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 (Cat. No. MA1-21315) at 1 µg/mL, followed by incubation with Goat anti-Mouse HRP Conjugate (Cat. No. 32430) at 100 ng/mL. SuperSignal West Pico PLUS substrate (Cat. No. 34577) was used for detection.

Robust signal

Robust signal

Robust signal with SuperSignal West Pico PLUS substrate. Detection of the indicated targets was performed using 2-fold serial dilutions of HEK293 or HeLa cell lysates, starting at 4 µg/well or 20 µg/well, respectively. Following separation by SDS-PAGE, proteins were transferred with Invitrogen Power Blotter (Cat. No. PB0013). The membranes were blocked with 5% nonfat dry milk dissolved in Pierce 20X TBS Tween 20 Buffer (Cat. No. 28360), and incubated with Invitrogen antibodies against beta-Catenin (Cat. No. MA1-300), STAT3 (Cat. No. MA1-13042), or WNT1 (Cat. No. MA5-15544) followed by incubation with Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP Conjugate (Cat. No. 31430) 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.

SuperSignal West Dura

Thermo Scientific SuperSignal West Dura Extended Duration Substrate for HRP is optimized for high sensitivity, wide dynamic range and long signal duration, making it ideal for sensitive quantitative western blots. Unlike substrates with signals that decline to barely detectable levels in 30–60 minutes, the signal produced with SuperSignal West Dura chemiluminescent substrate is stable for 24 hours.

Quantitative western blots for a range of target protein abundance

Quantitative western blot analysis for low, medium and high abundant proteins using SuperSignal West Dura ECL kit

Obtain quantitative western blots with SuperSignal West Dura Substrate. A 3-fold serial dilution of A431 or A549 lysate was loaded onto a Bolt 4-12% Bis-Tris-Plus gel (NW04122BOX) and electrophoresed at 200V for 20 minutes. Gels were transferred to nitrocellulose membranes using the iBlot 2 Transfer Device (P0, 7 min). Blots where processed using the Bandmate Automated Western Blot Processor. Membranes were blocked in 1X Clear Milk Blocking Buffer (Cat. No. 37587). Each membrane was probed with a single primary antibody diluted in 1X Clear milk. Mouse anti-p23 (1:25,000) (Cat. No. MA3-414), rabbit anti-Hsp90 (1:25,000) (Cat. No. PA3-013), mouse anti-Pan Ras10 (1:5,000) (Cat. No. MA1-012) and mouse anti-Mu-Calpain (1:5,000) (Cat. No. MA3-940), followed by an incubation with secondary antibodies Stabilized Goat anti-Rabbit HRP 10µg/mL (1:500) (Cat. No. 32460) or Stabilized Goat anti-Mouse HRP 10 µg/mL (1:5 00) (Cat. No. 32430). SuperSignal West Dura substrate (Cat. No. 34076) was used for detection.

Mid-femtogram detection

Obtain mid-femtogram detection with SuperSignal West Dura substrate

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. Seven 1:1 serial dilutions were then prepared and applied at 10 μL/well. After electrophoresis, proteins were transferred to PVDF membrane (Cat. No. 88518), and then the membrane was blocked with SuperBlock T20 (TBS) Blocking Buffer (Cat. No. 37536). The membrane was incubated with biotinylated anti-His antibody (Cat. No. MA1-21315-BTIN) at 1 μg/mL and then with Invitrogen Streptavidin, HRP Conjugate (Cat. No. 21126) at 1:50,000 dilution. SuperSignal West Dura substrate (Cat. No. 34076) was used for detection.

Better sensitivity and signal duration

Better sensitivity and signal duration with SuperSignal West Dura substrate

Better sensitivity and signal duration with SuperSignal West Dura substrate. A431 cell lysate was diluted in electrophoresis reducing sample buffer. Lane 1 contained 5 µg of A431 lysate. Five 1:1 serial dilutions were then prepared and applied at 10 μL/well. After electrophoresis, proteins were transferred to a nitrocellulose membrane using the Invitrogen Power Blotter (Cat. No. PB0013). Membranes were blocked with 5% milk in TBST Buffer. The membranes were incubated with Anti-beta-Catenin (Cat. No. MA1-300) at 0.3 μg/mL and then with Goat anti-Mouse HRP Conjugate (Cat. No. 32430) at 20 ng/mL. Identical blots were incubated in either SuperSignal West Dura substrate (Cat. No. 34076), Clarity™ Western ECL Blotting Substrate (Bio-Rad Laboratories, Inc.) or Immobilon™ Western Chemiluminescent HRP Substrate (EMD Millipore Corp.) according to respective manufacturer's instructions. 30-second exposures of the resulting blots were acquired.

SuperSignal West Atto

SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate is an ultrasensitive enhanced chemiluminescent (ECL) substrate that enables high attogram level protein detection by western blot analysis with the horseradish peroxidase (HRP) enzyme. It provides the highest-level sensitivity and better signal-to-noise ratios than other commercially available high-performance HRP substrates. It is the ideal choice for detection of very low-abundance targets or when using precious samples that require maximum levels of sensitivity.

Detect lower levels of protein

Figure 1. Chemiluminescent substrate product comparison with SuperSignal West Atto substrate.

  1. Akt (pan) detection in HeLa lysate was performed using HRP chemiluminescent substrates, SuperSignal West Atto, Bio-Rad Clarity Max, Amersham ECL Select, Millipore Immobiolon Western, Bio-Rad Clarity and standard ECL. The blots were developed using AKT (pan) monoclonal antibody (Cat. No. MA5-14916), followed by HRP-conjugated goat anti-rabbit IgG secondary antibody (Cat. No. 32460). Blots were incubated with respective substrates per product instructions. Images were captured using the iBright Imaging System.
  2. The signal-to-noise ratios for the western blot bands in the boxed area for SuperSinal West Atto, Bio-Rad Clarity Max, Amersham ECL Select, Millipore Immobilon Western, Bio-Rad Clarity and ECL are shown in bar graph form. SuperSignal West Atto provided the best signal-to-noise ratios across all protein loads. ECL didn’t have detectable signal above background noise after 0.625ug. Clarity and Immobilon didn’t have detectable signal above background noise after 0.313ug.

Use less samples

SuperSignal West Atto substrate allows use of very dilute samples. p23 in whole cell HeLa cell lysates was detected with SuperSignal West Atto, Bio-Rad Clarity or Millipore Immobion. Only 1.25 µg of cell lysate was needed to get strong signal with SuperSignal West Atto, whereas 20 µg of cell lysate was required to get the same level of detection with the other two substrates.

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 (Cat. No. PB3210). The blots were incubated with mouse Anti p23 (Cat. No. MA3-414), follow by goat anti-mouse HRP conjugate (Cat. No. 32430). Finally, respective blots were incubated with SuperSignal West Atto Substrate (Part No. A38558), Clarity™ Western ECL Blotting Substrate (Bio-Rad Laboratories, Inc.) or Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore Corp.) per product instructions. All blots were simultaneously acquired using the iBright FL Imaging System.

Thermo Scientific substratePrimary benefitsSensitivitySignal duration (hours)Recommended antibody dilution
Pierce ECL PlusProduces both strong chemiluminescent and fluorescent signal Low picogram to mid-femtogram51°: 0.05–1 μg/mL
2°: 5–40 ng/mL
SuperSignal West FemtoExcellent sensitivity with optimized conditionsLow to mid-femtogram81°: 10–200 ng/mL
2°: 2–10 ng/mL

Customer story

"The Atto substrate has been working very well in our hands. We actually have a protein that we haven’t been able to detect… until we tried Atto! It is now a mainstay in the lab."

—Jackie Dillon, PhD, Senior Research Associate, Benson Hill, Inc.

Resources