Thermo Scientific SuperSignal West Femto 最高灵敏度底物是一款具有超高灵敏度的增强型化学发光(ECL)底物，可通过辣根过氧化物酶(HRP)实现低飞克级的免疫印迹检测。当SuperSignal West Femto底物与优化的抗体浓度和封闭缓冲液配合使用时，可检测到常规ECL底物无法检测的低丰度靶标蛋白。

NIH3T3 lysate was diluted in electrophoresis reducing sample buffer. Lane 1 contained 5µg of NIH3T3 lysate. Five 1:1 serial dilutions were then prepared and applied at 10μL/well. After electrophoresis, proteins were transferred to nitrocellulose membrane (Part No. 88018) using the Pierce G2 Fast Blotter (Part No. 62288) and Pierce 1-Step Transfer Buffer (Part No. 84731). Membranes were blocked with 5% milk in TBST Buffer. The membranes were incubated with Anti-Erk1 (Part No. MA1-13041) at 0.2μg/mL and then with Goat anti-Mouse Horseradish Peroxidase Conjugate (Part No. 32430) at 5ng/mL. Identical blots were incubated in either SuperSignal West Femto Substrate (Part No. 34096), Clarity™ ECL Western 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 10in. (Part No. 34091).

图2 Use less and see more with SuperSignal West Femto Substrate

A431cell lysate was diluted in electrophoresis reducing sample buffer at 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625µg/well with a 10µL/well load. After electrophoresis, proteins were transferred to nitrocellulose membrane (Part No. 88018) using the Pierce G2 Fast Blotter (Part No. 62288) and Pierce 1-Step Transfer Buffer (Part 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 (Part No. MA1-300) as indicated, followed by incubation with the appropriate dilution of Goat anti-Mouse Horseradish Peroxidase Conjugate (Part No. 31460). SuperSignal West Femto Substrate (Part No. 34095) was used for detection. Ten second exposures were acquired on CL-XPosure Film, 8 x 10 in. (Part No. 34091) and the myECL Imager (Part No. 62236). Exposures from the myECL Imager were inverted and contrasted (black=62,000, white=65,535, gamma=1.0).

图3 True femtogram levels of detection with SuperSignal West Femto Maximum Sensitivity Substrate

Purified IκB was serially diluted from 100 to 1fg and then electrophoresed on a 4-20% mini gel. The protein was transferred to PVDF membrane and blocked withStartingBlock Blocking Buffer for 1 hour at room temperature. The blot was incubated in rabbit anti-IκBα (1mg/mL) at 1:1000 dilution overnight at 4°C, followed by incubation in HRP-conjugated goat anti-Rabbit IgG (1mg/mL) at 1:200,000 dilution for 1 hour at room temperature. The membrane was exposed to CL-XPosure Film for 1 minute.