Western blot imaging solutions
Successful Western blot imaging requires a suitable protein detection technique in combination with an appropriate imaging device.
Researchers utilize X-ray film, digital imaging solutions, or both. Digital solutions include charge-coupled device (CCD) camera-based imagers and scanner-based systems. The choice of imaging solution depends on the protein detection method and the advantages and limitations of each solution.
When to use X-ray film for imaging Western blots
X-ray film is suitable for use with either radioactive or chemiluminescent detection.
Exposure to light imprints an image on X-ray film. The amount of light that reaches the film determines the image, with brighter, more intense light producing a stronger image. In Western blotting, this light comes from labeling proteins with chemiluminescent or radioactive signals.
In chemiluminescent labeling, horseradish peroxidase (HRP)-conjugated secondary antibodies react with enhanced chemiluminescence (ECL) reagents to emit light.
In radioactive labeling, autoradiography on photographic film converts emitted radiation into light, increasing the otherwise limited sensitivity of the radioisotope.
The intensity of imprint on an X-ray film is proportional to the signal. In turn, this signal relates to the amount of protein present on the Western blot membrane, which allows quantitation.
Advantages/limitations of X-ray film
Although use of autoradiography in Western blotting is decreasing with the emergence of new cost- and time-saving options, it is still highly sensitive. This technique also enables flexible exposure times, which can be helpful when trying to detect very weak signals.
A key limitation of film is in quantitative analysis. Capturing both strong and weak signals on a single film can lead to high-intensity signals saturating the film. This saturation narrows the linear dynamic range available for quantitation, making film better suited to confirm the presence or absence of a protein band.
Logistically, the use of X-ray film requires a dark room and developing chemicals that need careful handling and disposal.
When to use a CCD camera for imaging Western blots
CCD camera imaging is suitable for chemiluminescence and fluorescence detection.
This technique uses a light source to illuminate the membrane, when needed, or excite fluorophores at specific wavelengths. Lenses collect the resulting light from the imaging field, focusing it on a two-dimensional CCD array.
In fluorescence imaging, a broad-spectrum light source combined with filters enables users to select excitation wavelengths for producing an image from specific fluorophores.
The resulting digital image is essentially a pattern of charge proportional to the light from the chemiluminescence reaction or fluorescent dyes. The intensity of the image is proportional to the amount of light produced, indicating protein quantity.
Advantages/limitations of CCD camera-based imagers
CCD camera-based imagers provide high sensitivity and a broad linear dynamic range, making them suitable for precise quantitative imaging. The digital images are easy to analyze and share with accompanying software, to incorporate into presentations and papers, and to archive.
CCD-based cameras have a critical advantage over laser-based scanners for the detection of chemiluminescent emissions. In laser scanners, the light collecting scan head, moves over the sample at high speed and records data in less than a millisecond. A CCD-chip on the other hand can be exposed to chemiluminescent light from a fraction of a second to minutes to obtain the best possible data quality.
However, CCD-based cameras are sensitive to light, heat, and high-energy radiation, which can affect instrument performance and image quality. To combat quality issues, CCD cooling measures aim to reduce background noise and improve both sensitivity and linearity.
When to use a laser scanner for imaging Western blots
Scanner systems—often termed laser scanners as they use laser light for excitation—are most suitable for fluorescence detection. Although they can be used for chemiluminescent detection, this is not the preferred choice. Some scanner systems also support phosphorimaging for detection of radioactively labeled protein.
Scanners focus a narrow beam of monochromatic laser light on the membrane, collecting and filtering fluorophore-emitted light. A photomultiplier tube detects the photons and amplifies the signal reaching the detector, producing the digital image.
In single-channel or single-label experiments, emission filters allow a well-defined spectrum of emitted light to reach the detector. In multichannel or multilabel experiments, filter selection allows the scanning of different channels independently.
Digital images produced by laser scanners enable protein quantitation similar to X-ray film and CCD camera images, with the signals on the image being proportional to the light emitted from the fluorescent dyes.
Advantages/limitations of laser scanner-based systems
There are several advantages to using a laser scanner. The broad linearity of signal detection allows the user to quantitate high- and low-abundance proteins on the same membrane, with high resolution.
These scanners are also highly sensitive thanks to amplification of the fluorescence signal by laser excitation. In addition, these systems are easy to use, have autoscan functions, and enable high sample throughput.
Table 1 summarizes different imaging solutions, indicating their advantages and the compatible protein detection methods.
Table 1. Summary of Western blot imaging options and compatible detection methods
|Imaging option||Advantages||Compatible detection methods|
Flexible exposure times for detecting weak signals
|CCD camera-based imager||
Broad linear dynamic range
Digital image produced
|Laser scanner-based system||
Broad linear dynamic range
1 Recommended imaging solution for this protein detection method
2 Sensitivity is limited and not recommended for standard Western blotting
For more information on imaging options, please download the Imaging, Principles and Methods handbook.