b'SuperSignal West Dura Extended Duration Substrate Thermo Scientific SuperSignal West Dura Extended Duration Substrate is an enhanced chemiluminescence HRP substrate with stable light output for mid-femtogramlevel detection. It is ideal for quantitative western blotting.Features: 24-hour signal durationlight emission is stable for 10 times longer than with typical ECL substrates; acquire multiple exposures to obtain publication-quality blot images (Figures 28 and 29) Great sensitivityprovides detection of mid-femtogram levels of target proteins High intensityimmediate, stable signal generation provides easy detection via film or cooled CCD imaging systems Stable reagent24-hour working solution stability; one-year kit stability at room temperature Less antibody usageworks best with dilute antibody concentrationsPost- Chemiluminescent substrate productsubstrateSuperSignal West Dura Amersham ECL Prime 30 minutesChemiluminescent substrate product 1 minute4.5 hours Post-substrate Post-substrate 1 hourSuperSignal West Dura4 hoursClarity (Bio-Rad)Immobilon (Millipore) 20 hoursFigure 28. Better sensitivity and signal duration with SuperSignalFigure 29. Longer signal duration with SuperSignal West Dura West Dura Extended Duration Substrate. A431 cell lysate wasExtended Duration Substrate. Detection of Hsp86 in HeLa cell lysate serially diluted two-fold in electrophoresis reducing sample buffer andon nitrocellulose membranes. Lane 1 contained 10 g total protein separated by gel electrophoresis. Lane 1 contained 5 g of A431 lysate.(total 10 L/well). Two-fold serial dilutions were then prepared and After electrophoresis, proteins were transferred to a nitrocelluloseloaded at 10 L/well (lanes 25). After electrophoresis and transfer to membrane using the Power Blotter System and Pierce 1-Step Transfernitrocellulose membranes, blots were blocked with StartingBlock (TBS) Buffer. Membranes were blocked with 5% milk in TBST buffer. TheBlocking Buffer, probed with Invitrogen Rabbit Anti-Hsp86 Antibody at membranes were incubated with Invitrogen Mouse beta-Catenin Antibody1:2,000 dilution, followed by goat antirabbit IgG HRP secondary antibody at 0.3 g/mL and then with goat antimouse IgG, HRP conjugate, atat 6.6 ng/mL. Finally, respective blots were incubated with SuperSignal 20 ng/mL. Identical blots were incubated in either SuperSignal WestWest Dura substrate or Cytiva Amersham ECL Prime Western Blotting Dura substrate, Clarity Western ECL Substrate, or Immobilon WesternDetection Reagent per product instructions. At various time points Chemiluminescent HRP Substrate according to respective manufacturersfollowing substrate incubation, the two blots were imaged using a CCD instructions. Thirty-second exposures of the resulting blots werecamera imager. (Identical imaging exposure parameters were used for both simultaneously acquired on the MyECL Imager with the following settings:blots at each time point.)black = 65,296, white = 65,535, gamma = 1.0).49'