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Application Note 104WesternBrightTM Quantum Quantify chemiluminescent Western blots over a wide dynamic rangeWesternBright Quantum is a new chemiluminescent reagent specially formulated for CCD imaging. Thisnovel Horseradish peroxidase (HRP) substrate provides a strong, long-lasting signal, the broadest usefullinear range and high sensitivity for the most quantitative chemiluminescent Western assays.Chemiluminescence is the methodof choice for sensitive Westernblot detection, but has not beenconsidered quantitative, primarilybecause of the limited linear dynamicrange of film, and of commerciallyavailable substrates. WesternBrightQuantum HRP substrate overcomesthe limitations of other substrates,showing no substrate depletion athigh protein loads. WesternBrightQuantum is specially formulated for Figure 1. The principle of chemiluminescent Western blotting. A secondaryquantitative chemiluminescent antibody, conjugated to horseradish peroxidase (HRP) binds to a primaryWestern blotting, producing a linear antibody directed towards the protein of interest. The blot is incubated with a chemiluminescent substrate, which is converted by HRP into a light emittingsignal over a broad range of protein luminescent molecule.concentrations spanning 3 ordersof magnitude. Combined with CCD 72.9 pg 36.4 pg 18.2 pg 9.33 ng 4.66 ng 2.33 ng 1.16 ng 0.58 ng 0.29 ng 0.14 ng 9.1 pg 4.6 pg 2.3 pg 1.1 pg 0.6 pg 0.3 pg eimaging, which provides a much agreater linear dynamic range thanfilm, WesternBright Quantum allows bhighly quantitative data to beobtained from chemiluminescent cWestern blots. dThe broadest linear range for themost powerful quantitation Figure 2. Linear dynamic range of WesternBright Quantum. Identical Western blotsAccurate comparison of the intensities containing serial dilutions of transferrin were probed with a rabbit-anti-transferrinof different protein bands requires primary antibody, and a goat-anti-rabbit secondary antibody conjugatedthat the bands be within the linear to Horseradish peroxidase. The blots were incubated with chemiluminescentdynamic range of detection, which is substrates as recommended by each manufacturer. All blots were simultaneously imaged for 2 minutes on a CCD imager and all display parameters are identicalthe range of concentrations from the across all images shown in the figure. WesternBright Quantum shows thefaintest band that can be detected largest dynamic range out of all four substrates with the highest R2 value. a.to the most intense band for which Amersham™ ECL™ (GE Healthcare); b. Amersham™ ECL Plus™ (GE Healthcare);the signal is not saturated. When used c. SuperSignal® West Dura (Thermo Scientific); d. WesternBrights Quantum; e.to detect a Western blot containing Signal intensity vs protein amount plot showing best fit linear regression for all foura serial dilution of transferrin protein substrates. Bands that fall on the linear part of the curve are indicated. www.advansta.comPage 1 of 5
Application Note 104(Figure 2), WesternBright Quantum provides the broadest Substrate Linear Dynamic Rangelinear dynamic range when compared to several other WesternBright Quantum 4.6 pg – 4.7 ngcommercially available chemiluminescent substrates SuperSignal West Dura 9.1 pg – 2.3 ng(Figure 2e, Table 1). Notably, no substrate depletion isseen at any protein loads with WesternBright Quantum, ECL Plus 9.1 pg – 0.29 ngwhile substrate depletion interferes with detection ECL 73 pg – 1.2 ngof high amounts of protein by both ECL Plus and Table 1. Linear dynamic range for four chemiluminescentSuperSignal West Dura (seen as reverse intensity bands substrates. The linear dynamic range includes data pointsin Figures 2b and 2c). The ability to detect high amounts that produce the best possible linear regression fit (Figure 2e).of protein without substrate depletion contributes to the Analysis of the experiment depicted in Figure 2.increased dynamic range provided by WesternBrightQuantum. aFor determining linear range, best fit data analysiswas performed using linear regression. Data pointscorresponding to high protein amounts were excludedfrom datasets one by one as outliers to obtain thebroadest range with R2 value above 0.98. Using thismethod, WesternBright Quantum produced thebroadest linear range with the highest R2 value.Long lasting signalCCD exposure times with WesternBright Quantumare as quick as film exposures with other substrates.In addition, WesternBright Quantum provides greatersignal stability than the competition, allowing long-termCCD camera exposures to be conducted, if desired, b 5 min c 60 min d 10 hrto detect faint bands and low-abundance proteins.The long-lasting signal also means there is no need torush to image a blot, since the signal will remain strongfor several hours. To follow signal strength over time,GAPDH was detected on quadruplicate Western blots Figure 3. Signal duration of WesternBright Quantum. Blotscontaining serial dilutions of HeLa cell lysate (Figure 3). detected using WesternBright Quantum and three otherA band which was determined to be within the linear chemiluminescent substrates were imaged simultaneouslyrange of all four chemiluminescent substrates tested at several times over a 10 hour period. Each exposure was 2 min long for all blots and all substrates. Intensities of bandswas quantified at several time points over the next containing the same amount of protein are plotted for each10 hours. WesternBright Quantum maintained signal substrate in 3a. Images of this band on the WesternBrightstrength, with signal declining only approximately 30% Quantum blot obtained after 5 min (3b), 60 min (3c) and 10over a 60 minute span, while the signal from ECL, ECL hr (3d) are shown.Plus and SuperSignal West Dura each decayed byalmost 90%, to barely detectable levels, over the sameperiod (Figure 3a). In fact, the WesternBright Quantumsignal is still detectable in a 2 minute exposure 10 hourslater (inset, Figure 3a and 3d). www.advansta.comPage 2 of 5
Application Note 104Low background for high sensitivity a b c dWesternBright Quantum produces an exceptionallystrong signal with little to no background, for highsignal to noise ratio and excellent sensitivity. The lowbackground is especially apparent when WesternBrightQuantum is is used with film detection. To demonstratethe relative lack of background with WesternBrightQuantum compared to other chemiluminescentsubstrates, quadruplicate Western blots were detected Figure 4. Extremely low background with WesternBrightusing various HRP substrates as described in Methods. Quantum. Duplicate Western blots were developed usingThe four blots were simultaneously exposed to a single (a) ECL, (b) ECL Plus, (c) SuperSignal West Dura, or (d)film. After a 20 minute exposure, the blot detected using WesternBright Quantum as substrates. After a 20 minute exposure to film, WesternBright Quantum displays the bestWesternBright Quantum remains clear of background combination of sensitivity and signal with low background. All(Figure 4d) compared to ECL Plus (Figure 4b) or display parameters are identical across all images shown inSuperSignal West Dura (Figure 4c). this figure.Figure 5 demonstrates the superior sensitivity ofWesternBright Quantum on film; duplicate blots were a b cdetected using WesternBright Quantum or SuperSignalWest Pico. In a short exposure, WesternBright Quantum(Figure 5a) detects a band of a truncated protein notvisible with the other reagent (Figure 5b) unless a longerexposure is used (Figure 5c). WesternBright SuperSignal SuperSignalAccurately quantify low and high abundance Quantum West Pico West Picoproteins 45 sec exposure 45 sec exposure 15 min exposure Figure 5. Superior sensitivity of WesternBright Quantum withWesternBright Quantums high sensitivity, low film detection. WesternBright Quantum (a) or SuperSignalbackground and broad linear range combine to West Pico (Thermo Scientific) (b, c) were used to detectallow the accurate quantitation of low and high duplicate Western blots. A band (arrow in panel a) is detectedabundance proteins in a single experiment. Low by WesternBright Quantum in a brief exposure, while a muchabundance proteins can be detected due to the longer exposure is needed to detect the same band with SuperSignal West Pico (c).excellent sensitivity, and the broad linear dynamicrange allows high abundance proteins to be detectedwith the same exposure, without saturation of signal.Figure 6 demonstrates the superior performance ofWesternBright Quantum when probing for either a highabundance (actin) or low abundance (STAT-1) protein.Serial dilutions of extracts from A431 cells were assayedby replicate Western blots, probed for actin and STAT-1, and visualized using WesternBright Quantum or othercommercially available chemiluminescent substratesas described in Methods. For quantitative detectionon the linear part of the intensity vs protein curve,WesternBright Quantum proved to be 8-times more www.advansta.comPage 3 of 5
Application Note 104sensitive than ECL Plus in detecting STAT-1 (Figure 6g), actin STAT-1 5.00 µg 2.50 µg 1.25 µg 5.00 µg 2.50 µg 1.25 µgand twice as sensitive as ECL in detecting actin (Figure 625 ng 313 ng 156 ng 625 ng 313 ng 156 ng 78 ng 78 ng6c). a dSimply the best choice for CCD imaging ECL eWesternBright Quantum outperforms other enhancedchemiluminescent reagents, providing superior ECL Plussensitivity, signal duration, and linear dynamic range. b fSpecially formulated for CCD imaging, WesternBrightQuantum maintains a detectable signal in a two-minute WesternBright Quantumexposure for at least 10 hours, long after the signals from c gother substrates have decayed to levels undetectablewithout exposures many times longer.MethodsGel electrophoresisProteins were separated on self-made 12 %polyacrylamide gels using a Laemmli buffer system. Figure 6. Detect high and low abundance proteins withGels were 10 cm wide and 0.8 mm thick. WesternBright Quantum. Identical blots containing serialTransfer dilutions of cell A431 lysates were probed with a mixture orProteins were transferred to PVDF membrane using a primary antibodies to actin and STAT-1. The blots were then detected with WesternBright Quantum (panels b and f) ortank (Idea Scientific) and buffers developed by Bolt one of two other chemiluminescent substrates and imagedand Mahoney (1). Transfers were conducted for 25 min simultaneously with a 2 minute exposure using a CCD imager.at 24 V. The intensities of the bands were plotted against total protein loaded (panels c and g), demonstrating that WesternBrightBlocking, Primary and Secondary Antibodies, and Quantum provided the highest sensitivity and the broadestWashing linear dynamic range for both the high and low abundanceMembranes were blocked with 2% non-fat dry milk proteins.in AdvanWash buffer for 1 hour at room temperature(RT). Primary and secondary antibodies were diluted as Experiment Primary Dilution Secondary Dilutiondescribed in Table 2 in blocking buffer, and applied to antibody antibodymembranes for 1 hour at RT. Washing was conducted Linear Rabbit anti- 1:10,000 HRP-Goat- 1:20,000 dynamic transferrin anti-rabbitwith AdvanWash buffer according to the WesternBright range (Abcam, (Advansta, cat.user manual. cat. no. no. R-05072-500) ab122301)Substrate incubation Signal Mouse 1:2,000 HRP-Goat- 1:2,500 duration anti-GAPDH anti-mouseAfter washing, blots were incubated with (Millipore, (Advansta, cat.chemiluminescent substrates as recommended by cat. no. no. R-05071-500) MAB374)manufacturers. Incubation with WesternBright Quantum Cell lysates Mouse 1:1,000 HRP-Goat- 1:2,500substrate was done for 2 minutes. anti-actin anti-mouse (Sigma, cat. (Advansta, cat.Imaging no. A4700) no. R-05071-500)After incubation, blots were placed on a plastic Mouse 1:250 HRP-Goat- 1:2,500sheet, covered with Saran wrap, and imaged using a anti-STAT 1 anti-mouse (BD, cat. no. (Advansta, cat.FluorChem Q (Cell Biosciences). Images were analyzed 10116) no. R-05071-500)using AlphaView® software (Cell Biosciences). Table 2. Antibodies and dilutions used. www.advansta.comPage 4 of 5