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).