Chemiluminescent Substrates Performance Data
Thermo Scientific SuperSignal chemiluminescent HRP substrates offer excellent performance in western blotting applications, with longer light emission and stronger signal intensity than other luminol-based detection systems.
Several varieties of luminol-based chemiluminescent substrates are available, each designed to meet particular experiment needs.
| 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°: 0.2–10 μg/mL 2°: 0.07–1 μg/mL |
| Pierce ECL Plus | Same signal and lower price than competing ECL Plus substrates | Low picogram to mid-femtogram | 3 | 5 | 1°: 0.05–1 μg/mL 2°: 5–40 ng/mL |
| SuperSignal West Pico PLUS | Better signal and lower price than competing ECL substrates | Low picogram to high femtogram | 3 | 6 to 24 | 1°: 0.2–1 μg/mL 2°: 10–50 ng/mL |
| SuperSignal West Dura | Best for use with imaging equipment | Mid-femtogram | 4 | 24 | 1°: 0.02–1 μg/mL 2°: 4–20 ng/mL |
| SuperSignal West Femto | Most sensitive substrate for HRP detection | Low to mid-femtogram | 5 | 6 to 8 | 1°: 10–200 ng/mL 2°: 2–10 ng/mL |
| † Sensitivity Rating: 1=lowest, 5=highest | |||||
Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate offers better sensitivity, longer signal duration, and brighter intensity than our original SuperSignal West Pico Substrate. The intensity of light emission combined with exceptional duration allows for acquiring multiple exposures to effortlessly obtain publication-quality blot images.
Featured product data for SuperSignal West Pico PLUS 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.
Featured product data for SuperSignal West Pico PLUS substrateLow-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 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 to either PVDF (Cat. No. 88518) or nitrocellulose (Cat. No. 88018) membranes using the Pierce Power Blotter (Cat. No. 22834) and Pierce 1-Step Transfer Buffer (Cat. No. 84731). 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.
Thermo Scientific SuperSignal West Dura Extended Duration Substrate for HRP is optimized for high sensitivity and long signal duration, making it ideal for cooled-CCD camera-based detection systems. 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, allowing multiple film or camera exposures.
Featured product data for SuperSignal West Dura substrate
Longer signal duration with SuperSignal West Dura substrate. Detection of Hsp86 in HeLa cell lysate on nitrocellulose membranes. HeLa cell lysate was diluted in sample buffer and heated to 95˚C for 5 minutes. Lane 1 contained 10 μg total protein (total 10 μL/well). Four 1:1 serial dilutions were then prepared and loaded at 10 μL/well (Lanes 2–5). After transfer to nitrocellulose membranes (Cat. No. 88024), blots were blocked (Cat. No. 37542), probed with rabbit anti-Hsp 86 antibody (Cat. No. PA3-013) at 1:2,000 dilution, followed by goat anti-rabbit IgG HRP secondary antibody (Cat. No. 31460) at 6.6 ng/mL. Finally, respective blots were incubated with SuperSignal West Dura Substrate (Part No. 34076) or Amersham™ ECL™ Prime reagent (GE Healthcare, Cat. No. RPN2232) per product instructions. At various time-points following substrate incubation, the two blots were imaged using a CCD camera imager. (Identical imaging exposure parameters were used for both blots at each time-point.)
Mid-femtogram detection
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. 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 nitrocellulose membrane (Cat. No. 88018) using the Pierce Power Blotter (Cat. No. 22834) and Pierce 1-Step Transfer Buffer (Cat. No. 84731). 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 simultaneously acquired on the myECL Imager (Cat. No. 62236) with the following settings: black = 65,296, white = 65,535, and gamma = 1.0.
Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate is designed for low-femtogram-level detection by western blot analysis. When combined with optimized antibody concentrations and blocking buffers, SuperSignal West Femto substrate enables detection of target proteins in amounts that are too small to be seen with typical ECL substrates.
Featured product data for SuperSignal West Femto substrate
True femtogram levels of detection with SuperSignal West Femto substrate. Purified IκB was serially diluted from 100 to 1 fg and then electrophoresed on a 4–20% mini gel. The protein was transferred to PVDF membrane and blocked with StartingBlock Blocking Buffer for 1 hour at room temperature. The blot was incubated in rabbit anti-IκBα (1 mg/mL) at 1:1,000 dilution overnight at 4°C, followed by incubation in HRP-conjugated goat anti-Rabbit IgG (1 mg/mL) at 1:200,000 dilution for 1 hour at room temperature. The membrane was exposed to Thermo Scientific CL-XPosure Film for 1 minute.
Maximum sensitivity and duration
Obtain maximum sensitivity and signal duration with SuperSignal West Femto substrate. NIH3T3 lysate was diluted in electrophoresis reducing sample buffer. Lane 1 contained 5 µg of NIH3T3 lysate. Five 1:1 serial dilutions were then prepared and applied at 10 μL/well. After electrophoresis, proteins were transferred to nitrocellulose membrane (Cat. No. 88018) using the Pierce Power Blotter (Cat. No. 22834) and Pierce 1-Step Transfer Buffer (Cat. No. 84731). Membranes were blocked with 5% milk in TBST Buffer. The membranes were incubated with Anti-Erk1 (Cat. No. MA1-13041) at 0.2 μg/mL and then with Goat anti-Mouse HRP Conjugate (Cat. No. 32430) at 5 ng/mL. Identical blots were incubated in either SuperSignal West Femto substrate (Cat. No. 34096), Clarity™ Western ECL Blotting Substrate (Bio-Rad Laboratories, Inc.), Luminata™ Forte Western HRP substrate (EMD Millipore Corp.) or Immobilon™ Western Chemiluminescent HRP Substrate (EMD Millipore Corp.) according to each respective manufacturer's instructions. Two-minute exposures of the resulting blots were simultaneously acquired on a single CL-XPosure Film, 8 x 10 in. (Cat. No. 34091).
Save your antibodies
Use less and see more with SuperSignal West Femto substrate. A431 cell lysate was diluted in electrophoresis reducing sample buffer at 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 µg/well with a 10 µL/well load. After electrophoresis, proteins were transferred to nitrocellulose membrane (Cat. No. 88018) using the Pierce Power Blotter (Cat. No. 22834) and Pierce 1-Step Transfer Buffer (Cat. No. 84731). The membrane was blocked with 5% milk in TBST and cut into three strips to optimize antibody dilutions. Membranes were then incubated with anti beta-Catenin (Cat. No. MA1-300) as indicated, followed by incubation with the appropriate dilution of Goat anti-Mouse HRP Conjugate (Cat. No. 31460). SuperSignal West Femto substrate (Cat. No. 34095) was used for detection. Ten-second exposures were acquired on CL-XPosure Film, 8 x 10 in. (Cat. No. 34091) and the myECL Imager (Cat. No. 62236). Exposures from the myECL Imager were inverted and contrasted (black=62,000, white=65,535, gamma=1.0).
| 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°: 0.2–10 μg/mL 2°: 0.07–1 μg/mL |
| Pierce ECL Plus | Same signal and lower price than competing ECL Plus substrates | Low picogram to mid-femtogram | 3 | 5 | 1°: 0.05–1 μg/mL 2°: 5–40 ng/mL |
| SuperSignal West Pico PLUS | Better signal and lower price than competing ECL substrates | Low picogram to high femtogram | 3 | 6 to 24 | 1°: 0.2–1 μg/mL 2°: 10–50 ng/mL |
| SuperSignal West Dura | Best for use with imaging equipment | Mid-femtogram | 4 | 24 | 1°: 0.02–1 μg/mL 2°: 4–20 ng/mL |
| SuperSignal West Femto | Most sensitive substrate for HRP detection | Low to mid-femtogram | 5 | 6 to 8 | 1°: 10–200 ng/mL 2°: 2–10 ng/mL |
| † Sensitivity Rating: 1=lowest, 5=highest | |||||
Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate offers better sensitivity, longer signal duration, and brighter intensity than our original SuperSignal West Pico Substrate. The intensity of light emission combined with exceptional duration allows for acquiring multiple exposures to effortlessly obtain publication-quality blot images.
Featured product data for SuperSignal West Pico PLUS 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.
Featured product data for SuperSignal West Pico PLUS substrateLow-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 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 to either PVDF (Cat. No. 88518) or nitrocellulose (Cat. No. 88018) membranes using the Pierce Power Blotter (Cat. No. 22834) and Pierce 1-Step Transfer Buffer (Cat. No. 84731). 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.
Thermo Scientific SuperSignal West Dura Extended Duration Substrate for HRP is optimized for high sensitivity and long signal duration, making it ideal for cooled-CCD camera-based detection systems. 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, allowing multiple film or camera exposures.
Featured product data for SuperSignal West Dura substrate
Longer signal duration with SuperSignal West Dura substrate. Detection of Hsp86 in HeLa cell lysate on nitrocellulose membranes. HeLa cell lysate was diluted in sample buffer and heated to 95˚C for 5 minutes. Lane 1 contained 10 μg total protein (total 10 μL/well). Four 1:1 serial dilutions were then prepared and loaded at 10 μL/well (Lanes 2–5). After transfer to nitrocellulose membranes (Cat. No. 88024), blots were blocked (Cat. No. 37542), probed with rabbit anti-Hsp 86 antibody (Cat. No. PA3-013) at 1:2,000 dilution, followed by goat anti-rabbit IgG HRP secondary antibody (Cat. No. 31460) at 6.6 ng/mL. Finally, respective blots were incubated with SuperSignal West Dura Substrate (Part No. 34076) or Amersham™ ECL™ Prime reagent (GE Healthcare, Cat. No. RPN2232) per product instructions. At various time-points following substrate incubation, the two blots were imaged using a CCD camera imager. (Identical imaging exposure parameters were used for both blots at each time-point.)
Mid-femtogram detection
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. 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 nitrocellulose membrane (Cat. No. 88018) using the Pierce Power Blotter (Cat. No. 22834) and Pierce 1-Step Transfer Buffer (Cat. No. 84731). 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 simultaneously acquired on the myECL Imager (Cat. No. 62236) with the following settings: black = 65,296, white = 65,535, and gamma = 1.0.
Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate is designed for low-femtogram-level detection by western blot analysis. When combined with optimized antibody concentrations and blocking buffers, SuperSignal West Femto substrate enables detection of target proteins in amounts that are too small to be seen with typical ECL substrates.
Featured product data for SuperSignal West Femto substrate
True femtogram levels of detection with SuperSignal West Femto substrate. Purified IκB was serially diluted from 100 to 1 fg and then electrophoresed on a 4–20% mini gel. The protein was transferred to PVDF membrane and blocked with StartingBlock Blocking Buffer for 1 hour at room temperature. The blot was incubated in rabbit anti-IκBα (1 mg/mL) at 1:1,000 dilution overnight at 4°C, followed by incubation in HRP-conjugated goat anti-Rabbit IgG (1 mg/mL) at 1:200,000 dilution for 1 hour at room temperature. The membrane was exposed to Thermo Scientific CL-XPosure Film for 1 minute.
Maximum sensitivity and duration
Obtain maximum sensitivity and signal duration with SuperSignal West Femto substrate. NIH3T3 lysate was diluted in electrophoresis reducing sample buffer. Lane 1 contained 5 µg of NIH3T3 lysate. Five 1:1 serial dilutions were then prepared and applied at 10 μL/well. After electrophoresis, proteins were transferred to nitrocellulose membrane (Cat. No. 88018) using the Pierce Power Blotter (Cat. No. 22834) and Pierce 1-Step Transfer Buffer (Cat. No. 84731). Membranes were blocked with 5% milk in TBST Buffer. The membranes were incubated with Anti-Erk1 (Cat. No. MA1-13041) at 0.2 μg/mL and then with Goat anti-Mouse HRP Conjugate (Cat. No. 32430) at 5 ng/mL. Identical blots were incubated in either SuperSignal West Femto substrate (Cat. No. 34096), Clarity™ Western ECL Blotting Substrate (Bio-Rad Laboratories, Inc.), Luminata™ Forte Western HRP substrate (EMD Millipore Corp.) or Immobilon™ Western Chemiluminescent HRP Substrate (EMD Millipore Corp.) according to each respective manufacturer's instructions. Two-minute exposures of the resulting blots were simultaneously acquired on a single CL-XPosure Film, 8 x 10 in. (Cat. No. 34091).
Save your antibodies
Use less and see more with SuperSignal West Femto substrate. A431 cell lysate was diluted in electrophoresis reducing sample buffer at 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 µg/well with a 10 µL/well load. After electrophoresis, proteins were transferred to nitrocellulose membrane (Cat. No. 88018) using the Pierce Power Blotter (Cat. No. 22834) and Pierce 1-Step Transfer Buffer (Cat. No. 84731). The membrane was blocked with 5% milk in TBST and cut into three strips to optimize antibody dilutions. Membranes were then incubated with anti beta-Catenin (Cat. No. MA1-300) as indicated, followed by incubation with the appropriate dilution of Goat anti-Mouse HRP Conjugate (Cat. No. 31460). SuperSignal West Femto substrate (Cat. No. 34095) was used for detection. Ten-second exposures were acquired on CL-XPosure Film, 8 x 10 in. (Cat. No. 34091) and the myECL Imager (Cat. No. 62236). Exposures from the myECL Imager were inverted and contrasted (black=62,000, white=65,535, gamma=1.0).
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