Western Blotting Using Enhanced Chemiluminescence
Choose the perfect substrate for your needs.
by Thermo Scientific Pierce Protein Biology Team - 06/01/11
Chemiluminescence is one of the most popular methods of detection in western blotting applications because it offers several advantages over precipitating substrates, including high sensitivity, high signal-to-noise ratios, and the ability to strip and reprobe the blot. Additionally, chemiluminescent detection can be quantitative when blots are analyzed using CCD-based imagers.1 Among chemiluminescent substrates, the luminol-based substrates that produce light in the presence of horseradish peroxidase (HRP) and peroxide are the most commonly used.
Thermo Scientific Pierce Chemiluminescent HRP Substrates provide a range of convenient and reliable solutions for western blot analysis that vary in levels of detection sensitivity and signal duration (Table 1). Thermo Scientific SuperSignal Substrates produce accurate, consistent and robust results with better sensitivity and longer signal duration than other commercially available substrates. An economical chemiluminescent substrate for routine Western blotting is the Thermo Scientific Pierce ECL Substrate.
Signal duration and detection sensitivity of popular chemiluminescent western blotting substrates were analyzed in multiple detection platforms and different target proteins. Performance of the Pierce Substrates was compared to the Luminata Substrates (EMD Millipore). The two parameters analyzed are significant contributors to data quality from western blots.
We compared the signal duration of Pierce ECL Substrate to Luminata Classico Substrate (EMD Millipore). The Pierce ECL Substrate produced a much stronger signal, which continued 1 hour after substrate application; however, the signal generated using the Luminata Classico Substrate completely faded after 1 hour (Figure 1). Similarly, signal duration using SuperSignal West Dura Substrate was compared to Luminata Crescendo Substrate. The signal from the Luminata Crescendo Substrate substantially weakened 2 hours after substrate incubation. In contrast, there was sustained light output from SuperSignal West Dura Substrate (Figure 2).
Figure 1. Thermo Scientific Pierce ECL Western Blotting Substrate provides better sensitivity and longer lasting signal than a substrate from another supplier. K562 cell lysate was diluted in Laemmli sample buffer and heated to 95˚C for 5 minutes. Lane 1 contained 10μg total protein and four 1:1 serial dilutions were prepared and applied to Lanes 2-5. After electrophoresis, proteins were transferred to nitrocellulose membrane (Part No. 88013), and 5% milk prepared in TBST was used for blocking. The membranes were probed with mouse anti-ERK 1 (Ab No. MA1-13041) diluted 1:1000 and goat anti-mouse HRP antibody (Part No. 31430) at 100ng/mL. Pierce ECL Chemiluminescent Substrate (Part No. 32106) or Luminata Classico Substrate (EMD Millipore) was used for detection per manufacturer’s guidelines. Blots were exposed to Thermo Scientific CL-XPosure Film (Part No. 34091) for 1 minute (1-minute film exposure) and 1 hour (20-minute film exposure) after substrate incubation.
Figure 2. Thermo Scientific SuperSignal West Dura Substrate outperforms a similar substrate from another supplier. HeLa cell lysate was diluted in Laemmli sample buffer and heated to 95˚C for 5 minutes. Lane 1 contained 10μg total protein and four 1:1 serial dilutions were prepared and applied to Lanes 2-5. After electrophoresis, proteins were transferred to nitrocellulose membranes. Thermo Scientific StartingBlock in TBS Blocking Buffer (Part No. 37542) was used for blocking. The membranes were probed with rabbit anti-β-catenin antibody (Ab No. PA5-16429) diluted 1:2000 and goat anti-rabbit HRP antibody (Part No. 31460) at 4ng/mL. SuperSignal West Dura Substrate or Luminata Crescendo Substrate (EMD Millipore) was used for detection per manufacturer’s guidelines. The blots were exposed to X-ray film (Part No. 34091) 10 minutes and 2 hours after substrate incubation (5-minute film exposure).
The SuperSignal West Dura Substrate is optimized for both high sensitivity and long signal duration, making it ideal for cooled-CCD camera-based detection systems. Signal duration of SuperSignal West Dura Substrate was compared to Amersham ECL Prime Substrate (GE Healthcare Life Sciences). Blots were analyzed using a CCD camera, and the light output was quantitated at 30 minutes, 1 hour, 4 hours and 20 hours after substrate incubation. The SuperSignal West Dura Substrate sustained light output 20 hours after incubation (Figure 3).
Because of the scarcity and high costs of some primary antibodies, an ultra-sensitive substrate that enables the use of low antibody concentrations is desirable. SuperSignal West Femto Maximum Sensitivity Substrate was able to produce a detectable signal when used with the primary antibody at 0.05μg/mL (1:20,000 dilution from 1mg/mL) and the secondary antibody at 2.5ng/mL (Figure 4).
- Signal duration of the Pierce Substrates was much longer than the corresponding Luminata and ECL Prime Substrates.
- Detection sensitivity of the Pierce Substrates was greater than the corresponding Luminata Substrates.
- SuperSignal West Femto Maximum Sensitivity Substrate enables the use of low antibody concentrations without sacrificing sensitivity.
Total proteins in cell lysates were separated by electrophoresis and transferred to a nitrocellulose membrane. Membranes were incubated in blocking buffer for 1 hour at room temperature or overnight at 4°C. The primary antibody was diluted in blocking buffer and incubated on the membrane for 1 hour at room temperature. HRP-conjugated secondary antibody conjugate was diluted in blocking buffer. Membranes were washed and incubated with the secondary antibody conjugate for 30 minutes at room temperature.
- Alegria-Schaffer, A., et al. (2009). Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Methods Enzymol 463:573-99.
For Research Use Only. Not for use in diagnostic procedures.