b'SuperSignal West Pico PLUS Chemiluminescent SubstrateThermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate is an enhanced chemiluminescent HRP substrate that enables low-picogram to high-femtogram levels of protein detection by western blot analysis. The intensity of the light emission combined with the exceptional duration allows for the acquisition of multiple exposures to more easily obtain publication-quality blot images. SuperSignal West Pico PLUS substrate is compatible with numerous membranes, blocking reagents, and a wide range of antibody dilutions, making it an ideal choice for most westernSuperSignalWest Pico PLUSblotting applications (Figures 2527). Bio-Rad ClarityTarget: beta-Catenin elF4E RSK2 EzrinSuperSignal West Pico PLUS ChemiluminescentMW: 85 kDa 25 kDa 80 kDa 69 kDaSubstrate characteristics: Lysate (amt): HEK (4 g) HEK (4 g) HeLa (20 g) HeLa (20 g)Membrane: PVDF PVDF Nitrocellulose Nitrocellulose Sensitivity: pictogram to femtogram sensitivity Figure 26. Performance of SuperSignal West Pico PLUS substrate compared to another chemiluminescent substrate. Detection of theStability: 8 hr working solution stability; 1-year kitindicated targets was performed using 2-fold serial dilutions of HEK293 stability at room temperature or HeLa cell lysates, starting with the amount indicated in parentheses above. Following separation by SDS-PAGE, proteins were transferredCompatibility: nitrocellulose and PVDF membranes to either PVDF or nitrocellulose membranes using the Power Blotter System and Pierce 1-Step Transfer Buffer. The membranes were blocked with 5% nonfat dry milk dissolved in Thermo Scientific Pierce 20X TBSSignal duration: 624 hours Tween 20 Buffer, and incubated with antibodies against beta-catenin, eIF4E, RSK2, or ezrin, followed by incubation with an Invitrogen goat anti Recommended primary antibody concentration:mouse IgG (H+L) secondary antibody (HRP conjugate), at a concentration 1:1,0001:5,000 dilution (0.21.0 g/mL) of 20 ng/mL. Chemiluminescent detection and substrate comparison was performed following a 5-minute incubation with either SuperSignal WestRecommended secondary antibody concentration:Pico PLUS or Bio-Rad Clarity substrates. Signal was captured using film.1:20,000100,000 dilution (1050 ng/mL)Initial10 5 2.5 1.25 0.625 0.3125 0.156 0.078 pg/well 1 hr2 hr3 hrbeta-Catenin STAT3 WNT1Figure 27. SuperSignal West Pico PLUS substrate produces robust B55,000 W65,535 G1Figure 25. SuperSignal West Pico PLUS provides low-picogramsignals over a long duration of time. Detection of the indicated targets to high-femtogram detection. TurboGFP-His-HA-Flag was dilutedwas performed using 2-fold serial dilutions of HEK293 or HeLa cell lysates, in electrophoresis reducing sample buffer. Lane 1 contained 10 pg ofstarting at 4 g/well or 20 g/well, respectively. Following separation by the purified protein with serial dilutions prepared 1:1 and applied atSDS-PAGE, proteins were transferred to either Thermo Scientific PVDF 10 L/well. After electrophoresis, proteins were transferred to nitrocelluloseor nitrocellulose membranes using the Power Blotter System and Pierce membrane using the Power Blotter System and Pierce 1-Step Transfer1-Step Transfer Buffer. The membranes were blocked with 5% nonfat dry Buffer. The membrane was blocked with SuperBlock Blocking Buffer.milk dissolved in Pierce 20X TBS Tween 20 Buffer, and incubated with The membrane was then incubated with anti-His antibody at 1 g/mL,Invitrogen antibodies against beta-catenin, STAT3, or WNT1 followed followed by incubation with HRP-conjugated goat antimouse IgGby incubation with goat antimouse IgG (H+L) secondary antibody at 100 ng/mL. SuperSignal West Pico PLUS substrate was used for(HRP conjugate) at a concentration of 20 ng/mL. Chemiluminescent detection. A 30-second exposure was acquired on the Thermo Scientificdetection was performed following a 5-minute incubation with SuperSignal MyECLImager, and the image was inverted and contrastedWest Pico PLUS substrate. Signal was captured on film at the indicated (black = 55,000, white = 65,535, gamma = 1.0). time points after addition of substrate.48'