Chemiluminescent Western blotting is widely used for protein detection. The chemiluminescent signal is usually detected and recorded by exposing the membrane to X-ray film.
With the development of digital imaging systems that employ charged-coupled device (CCD) cameras that capture the luminescent signals from Western blots, users now have many more options, such as greater sensitivity and higher dynamic range, for the detection and quantification of Western blots.
There has been a recent trend towards chemifluorescence, which gives users a number of benefits such as shorter exposure times and stable signal for weeks.
This article compares the performance of Syngene’s G:BOX Chemi IR6 imaging system with X-ray film for the visualization and quantification of chemifluorescence and chemiluminescent signals on Western blots.
Materials and methods
Precast Invitrogen gels (4-12% Bis-Tris, 12 well, 1 mm). MOPS was used as a running buffer. HeLa cell lysate (2.92 mg/ml sample) was mounted onto the precast gel. BioRad Precision All Blue was used as the marker.
Semi-wet Invitrogen Sure-Lock system was used for transfer. Also used were a nitrocellulose membrane (Invitrogen) and transfer buffer (50 ml 20x NuPAGE transfer buffer, 100 ml methanol and 850 MilliQ water).
Anti-actin monoclonal (1:5000) (Abcam, UK) was used as the primary antibody and Anti-mouse HRP (1:10,000) (Vector Laboratories, UK) as the secondary antibody.
Chemiluminescence imaging system
Syngene’s G:BOX Chemi IR6 system and a darkroom.
HeLa cell lysate was diluted in a ratio of 1:2, ranging from 29.2 to 0.028 µg/well, with 10 µl of each concentration loaded onto the gel.
After loading the gel with 2 µl of BioRad Precision All Blue marker, it was allowed to run for 30 minutes at 60 V. Then, the Sure-Lock system (Invitrogen, UK) was used to transfer the gel at 125 V for ~1 hour and 40 minutes. The blot was blocked for 1 hour in Odyssey block buffer.
Next, the membrane was incubated overnight with the Anti-actin antibody at 4 °C on a rocker. Following the incubation period, the membrane was washed 4 times for 5 minutes each with PBS and 0.1% Tween (PBST).
The membrane was incubated with the Anti-mouse HRP antibody in the dark, at room temperature for 1 hour, and then washed 4 times for 5 minutes, each with PBST. It was then incubated for 5 minutes with the chemiluminescent reagent ECL Plus (GE Healthcare, UK). The G:BOX Chemi IR6 imaging system was used to capture the image of the blot, which was then exposed to film.
The ECL Plus substrate can also be employed as a chemifluorescent reagent. Table 1 shows the proposed combinations of lighting and filter used for imaging the ECL Plus.
Table 1. Recommended lighting and filter combinations for visualizing ECL Plus on an imaging system
ECL Plus (Chemi)
ECL Plus (Fluorescence)
Figure 1 shows the comparison of three different methods of visualizing an ECL Plus Western blot (chemifluorescence, chemiluminescence, and X-ray film).
The results, as shown in Figure 1, reveal that all three methods possess the same sensitivity and exhibit a detection limit of 0.11µg.
The square of the correlation coefficient (R2) determines the linearity of the data. The GeneTools analysis software (Syngene, UK) was used to analyze the images of each blot. The intensities of the signal were plotted against the quantity of protein loaded onto the gel. Then, a linear curve fit was carried out (the graphs are shown below the matching blot images in Figure 1).
Both ECL Plus fluorescence and ECL Plus chemiluminescence had a linear detection, ranging from 1.83 to 0.11µg, plus a R2 value of 0.983 (Figure 1a) and 0.982 (Figure 1b), respectively.
ECL Plus exposed to the X-ray film had a R2 value of 0.905 (Figure 1c) and a dynamic range from 0.91 to 0.11µg, which is less than that of chemifluorescence and chemiluminescence methods.
Figure 1. Comparison of ECL Plus chemiluminescence, ECL Plus fluorescence and X-ray film HeLa whole cell lysate diluted 2-fold starting at 29.2µg. Blotted on to nitrocellulose membrane and detected with A) ECL Plus chemiluminescence, B) ECL Plus fluorescence and C) ECL Plus X-ray film. Imaged blots (top) and linearity plots based on analyzed data in GeneTools software (below). The dynamic range and R2 values are shown in the table.
X-ray film is typically employed to visually assess the relative band intensities. Such assessments are adequate in those situations where the comparison of signal intensities vary to a large extent. Yet, this method has certain limitations and therefore is not suitable for precise quantification of signals.
In certain cases, the signal on X-ray film allows the film to be quickly exposed to saturation, which results in strong dark bands on the film. This creates the perception that X-ray films are indeed more sensitive when compared to digital imaging. On the other hand, a greater dynamic range is seen in a CCD-captured image that will have the data distributed over a much wider range.
Therefore, moderate signals, which would have reached saturation (strong black bands) on the film, must lie in the middle (grey) range of the imaging system’s dynamic range. This process results in a more quantitative data.
Cost savings with digital imaging
The costs related to the use of X-ray film, such as the cost of film, the purchase and maintenance of film processors, the chemicals required to process the films, and the waste disposal of chemicals, can be prevented by using a digital imaging system.
Although, an initial investment cost is involved in the digital imaging systems, the reduced operational costs over time can make these systems a less expensive option.
Digital imaging systems offer a practical and easy solution for imaging and executing quantitative analysis of chemiluminescent blots. The G:BOX Chemi IR6 system from Syngene has a wider dynamic range and better linearity compared to X-ray film – 1.2 orders of magnitude (over the data range shown) and R2 of 0.9831 compared to 0.9 and R2 of 0.95, respectively.
The G:BOX Chemi systems enable easy set-up, high sensitivity, and a wide dynamic range, in addition to saving costs in the long run.
Produced from materials originally authored by Dr Lindsey Bunn, Applications Specialist, Syngene, A Division of Synoptics Ltd, Beacon House, Nuffield Road, Cambridge, CB4 1TF, UK
Syngene are a division of the Synoptics Group, founded in 1985 by imaging experts from the University of Cambridge. At Syngene we live and breathe image analysis because products specifically for gel documentation and fluorescence/chemiluminescence imaging are all we’ve ever focused on developing.
Our other divisions in the Synoptics Group, Synbiosis and Syncroscopy, develop imaging solutions for microbial and microscopy applications so we are complete life science imaging specialists. Synoptics Health focuses on imaging techniques within the clinical environment.
We are headquartered in Cambridge, a thriving scientific hub in the UK and have a centrally located US subsidiary in Frederick, USA. Globally, our products are supported by an international network of over 60 highly-trained distributors, all of whom employ imaging specialists.
Our world-leading technology includes a wide range of equipment for instant gel documentation, automated chemiluminescence imaging and TLC plate reading, which comply with current regulations specified by accreditation bodies and regulatory agencies.
Our systems are used globally by more than 75,000 scientists and you’ll find them successfully contributing accurate data to important projects in many of the world’s top pharmaceutical companies and major research institutes.
Syngene is a division of Synoptics Ltd. Synoptics Ltd is a company registered in England with company number 1874861. Its registered office address is Beacon House, Nuffield Road, Cambridge CB4 1TF. Synoptics Ltd VAT number is GB665 523522.
Synoptics Limited is fully compliant to the Waste Electrical & Electronic Equipment (WEEE) Regulations. We are a Member of the B2B Compliance scheme, which will handle our WEEE obligations on our behalf. Our Product Registration Number is WEE/AJ0049TZ. For further information, please contact B2B Compliance on: +44 (0) 1691 676 124.
Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.