WesternBright™ Quantum- Quantify chemiluminescent Western blots over a wide dynamic range
WesternBright Quantum is a new chemiluminescent reagent specially formulated for CCD imaging. This novel Horseradish peroxidase (HRP) substrate provides a strong, long-lasting signal, the broadest useful linear range and high sensitivity for the most quantitative chemiluminescent Western assays.
Chemiluminescence is the method of choice for sensitive Western blot detection, but has not been considered quantitative, primarily because of the limited linear dynamic range of film, and of commercially available substrates. WesternBright Quantum HRP substrate overcomes the limitations of other substrates, showing no substrate depletion at high protein loads. WesternBright Quantum is specially formulated for quantitative chemiluminescent Western blotting, producing a linear signal over a broad range of protein concentrations spanning 3 orders of magnitude. Combined with CCD imaging, which provides a much greater linear dynamic range than film, WesternBright Quantum allows highly quantitative data to be obtained from chemiluminescent Western blots.
The broadest linear range for the most powerful quantitation
Accurate comparison of the intensities of different protein bands requires that the bands be within the linear dynamic range of detection, which is the range of concentrations from the faintest band that can be detected to the most intense band for which the signal is not saturated. When used to detect a Western blot containing a serial dilution of transferrin protein (Figure 2), WesternBright Quantum provides the broadest linear dynamic range when compared to several other commercially available chemiluminescent substrates (Figure 2e, Table 1). Notably, no substrate depletion is seen at any protein loads with WesternBright Quantum, while substrate depletion interferes with detection of high amounts of protein by both ECL Plus and SuperSignal West Dura (seen as reverse intensity bands in Figures 2b and 2c). The ability to detect high amounts of protein without substrate depletion contributes to the increased dynamic range provided by WesternBright Quantum.
For determining linear range, best fit data analysis was performed using linear regression. Data points corresponding to high protein amounts were excluded from datasets one by one as outliers to obtain the broadest range with R2 value above 0.98. Using this method, WesternBright Quantum produced the broadest linear range with the highest R2 value.
Long lasting signal
CCD exposure times with WesternBright Quantum are as quick as film exposures with other substrates. In addition, WesternBright Quantum provides greater signal stability than the competition, allowing long-term CCD camera exposures to be conducted, if desired, to detect faint bands and low-abundance proteins. The long-lasting signal also means there is no need to rush to image a blot, since the signal will remain strong for several hours. To follow signal strength over time, GAPDH was detected on quadruplicate Western blots containing serial dilutions of HeLa cell lysate (Figure 3). A band which was determined to be within the linear range of all four chemiluminescent substrates tested was quantified at several time points over the next 10 hours. WesternBright Quantum maintained signal strength, with signal declining only approximately 30% over a 60 minute span, while the signal from ECL, ECL Plus and SuperSignal West Dura each decayed by almost 90%, to barely detectable levels, over the same period (Figure 3a). In fact, the WesternBright Quantum signal is still detectable in a 2 minute exposure 10 hours later (inset, Figure 3a and 3d).
Low background for high sensitivity
WesternBright Quantum produces an exceptionally strong signal with little to no background, for high signal to noise ratio and excellent sensitivity. The low background is especially apparent when WesternBright Quantum is is used with film detection. To demonstrate the relative lack of background with WesternBright Quantum compared to other chemiluminescent substrates, quadruplicate Western blots were detected using various HRP substrates as described in Methods. The four blots were simultaneously exposed to a single film. After a 20 minute exposure, the blot detected using WesternBright Quantum remains clear of background (Figure 4d) compared to ECL Plus (Figure 4b) or SuperSignal West Dura (Figure 4c).
Figure 5 demonstrates the superior sensitivity of WesternBright Quantum on film; duplicate blots were detected using WesternBright Quantum or SuperSignal West Pico. In a short exposure, WesternBright Quantum (Figure 5a) detects a band of a truncated protein not visible with the other reagent (Figure 5b) unless a longer exposure is used (Figure 5c).
Accurately quantify low and high abundance proteins
WesternBright Quantum's high sensitivity, low background and broad linear range combine to allow the accurate quantitation of low and high abundance proteins in a single experiment. Low abundance proteins can be detected due to the excellent sensitivity, and the broad linear dynamic range allows high abundance proteins to be detected with the same exposure, without saturation of signal. Figure 6 demonstrates the superior performance of WesternBright Quantum when probing for either a high abundance (actin) or low abundance (STAT-1) protein. Serial dilutions of extracts from A431 cells were assayed by replicate Western blots, probed for actin and STAT-1, and visualized using WesternBright Quantum or other commercially available chemiluminescent substrates as described in Methods. For quantitative detection on the linear part of the intensity vs protein curve, WesternBright Quantum proved to be 8-times more sensitive than ECL Plus in detecting STAT-1 (Figure 6g), and twice as sensitive as ECL in detecting actin (Figure 6c).
Simply the best choice for CCD imaging
WesternBright Quantum outperforms other enhanced chemiluminescent reagents, providing superior sensitivity, signal duration, and linear dynamic range. Specially formulated for CCD imaging, WesternBright Quantum maintains a detectable signal in a two-minute exposure for at least 10 hours, long after the signals from other substrates have decayed to levels undetectable without exposures many times longer.
Methods
Gel electrophoresis
Proteins were separated on self-made 12 % polyacrylamide gels using a Laemmli buffer system. Gels were 10 cm wide and 0.8 mm thick.
Transfer
Proteins were transferred to PVDF membrane using a tank (Idea Scientific) and buffers developed by Bolt and Mahoney (1). Transfers were conducted for 25 min at 24 V.
Blocking, Primary and Secondary Antibodies, and Washing
Membranes were blocked with 2% non-fat dry milk in AdvanWash buffer for 1 hour at room temperature (RT). Primary and secondary antibodies were diluted as described in Table 2 in blocking buffer, and applied to membranes for 1 hour at RT. Washing was conducted with AdvanWash buffer according to the WesternBright user manual.
Substrate incubation
After washing, blots were incubated with chemiluminescent substrates as recommended by manufacturers. Incubation with WesternBright Quantum substrate was done for 2 minutes.
Imaging
After incubation, blots were placed on a plastic sheet, covered with Saran wrap, and imaged using a FluorChem Q (Cell Biosciences). Images were analyzed using AlphaView® software (Cell Biosciences).
References
1. Bolt M.W. and Mahoney P.A. 1997. Anal Biochem. 247. 185-192
