SuperSignal Chemiluminescent Substrates

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.

Benefits you can expect from SuperSignal West Pico PLUS Chemiluminescent Substrate:

• Picogram to femtogram sensitivity—detect low-picogram to high-femtogram amounts of target protein on nitrocellulose or PVDF membranes
• High signal stability—incubated blots provide stable signal duration over the critical 4 hour time period, with up to 24 hours of light output under optimal conditions
• Exceptional robustness—provides high performance outside of the recommended antibody dilution ranges, including the most common 1:5,000 to 1:10,000 secondary antibody dilutions of a 1 mg/mL stock solution
• Stable reagent—8-hour working solution stability; 1-year kit stability at room temperature
• Economical—optimized for dilute antibody concentrations:
  • 0.2 to 1.0 µg/mL primary antibody (1:1,000 to 1:5,000 dilution from 1 mg/mL stock)
  • 10 to 50 ng/mL secondary antibody (1:20,000 to 1:100,000 dilution from 1 mg/mL stock)

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 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. A 30-second exposure was acquired on the Thermo Scientific™ myECL™ Imager (Cat. No. 62236). See product page for additional details.

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. Femtogram-level sensitivity with 24-hour light emission makes SuperSignal West Dura substrate the right choice for your western blot needs, and the right companion for your CCD imager.

Benefits you can expect from SuperSignal West Dura Extended Duration Substrate:

• 24-hour light emission—longer stable light emission than other enhanced chemiluminescent substrates for HRP; obtain multiple exposures to get publication-quality blots
• High sensitivity—with femtogram-level detection, see bands you’ve always had trouble viewing
• Strong intensity—immediate signal provides easy detection on film or compatible imaging system
• Stability—working solution is stable for at least 24 hours and the kit is stable for at least one year at room temperature
• Less antibody usage—works best with dilute antibody concentrations:
  • 20 ng to 1.0 µg/mL primary antibody (1:1,000 to 1:50,000 dilution from 1 mg/mL stock)
  • 4 to 20 ng/mL secondary antibody (1:50,000 to 1:250,000 dilution from 1 mg/mL stock)

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.)

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 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.

Benefits you can expect from SuperSignal West Femto Maximum Sensitivity Substrate:

• High sensitivity—detect low-femtogram (mid-zeptomole) amounts of protein in bands on nitrocellulose or PVDF membranes when probed with appropriate primary and secondary antibodies
• Quantitative—produces quantitative signal that is measurable over two orders of magnitude
• Intense signal—easy to capture an image by exposure to film or imaging system
• Excellent signal duration—8 hours of usable light output when conditions are optimized
• Stable reagent—kit components are stable for 1 year at 4°C or 6 months at room temperature
• Economical—optimized for extremely dilute antibody concentrations:
  • 1 ng to 0.2 µg/mL primary antibody (1:5,000 to 1:100,000 dilution from 1 mg/mL stock)
  • 2 to 10 ng/mL secondary antibody (1:100,000 to 1:500,000 dilution from 1 mg/mL stock)

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.

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).

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).

Instruments for chemiluminescence signal documentation. Instruments listed in the following table are capable of documenting signal from chemiluminescent substrates, such as the Thermo Scientific SuperSignal Substrates.