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Successful Western blot imaging requires a suitable protein detection technique in combination with an appropriate imaging device. Digital imagers provide high sensitivity and a broad linear dynamic range, making them applicable for precise quantitative imaging of gels and blots. The digital images are easy to analyze and share with accompanying software, to incorporate into presentations and papers, and to archive.

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.

Western blot imaging solutions

Chemiluminescence- and ECL-based methods are straightforward, necessitating only an HRP-conjugated antibody and a luminol reagent solution–both of which are readily available from commercial manufacturers. Visualization and imaging, whether using film (which is then digitally scanned) or camera-captured digital images, have relatively low technical requirements.

However, chemiluminescence does possess several limitations. First and foremost, it provides an indirect assessment of protein quantities. Additionally, signal saturation is a particular problem, especially for ubiquitously expressed housekeeping proteins, as each HRP bound to a secondary antibody has multiple binding sites for interaction with ECL substrates. This exponential signal amplification leads to a rapid plateau.

Fluorescence-based techniques for Western blotting were developed in response to these issues. Fluorophores offer a more direct assessment of protein abundance and greater sensitivity for both low and high protein amounts. Additionally, fluorescence-based methods facilitate multiplexing, allowing you to probe two or more proteins simultaneously. This means that you can avoid time-consuming stripping techniques which can potentially remove protein from the membrane, as well as concerns about signal overlap or over- or undersaturation. The latter is particularly useful when quantifying housekeeping proteins, which are likely many times more abundant than experimental proteins of interest.

Fluorescence vs chemiluminescence

Laser scanners have a scan head that passes over the gel or membrane section by section. The light is detected as the scan head moves to produce the individual pixels of the image. While, CCD camera-based imaging, on the other hand, works by detecting the entire sample at once, either with chemiluminescence or fluorescence. Varying the exposure time can improve detection or reduce noise.

Western blot imaging: CCD camera or laser scanner?

A key limitation of using X-ray 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.

In contrast, digital 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.

Western blot imaging solutions

Signal-to-noise optimization watch (SNOW) imaging mode is an automated imaging mode, used to detect more weak bands on Western blots and achieve high sensitivity without compromising image quality.

Image noise limits the range of signal intensities that can be analyzed accurately from an exposure. Poor resolution limits the viewing of fine details in the image, such as closely spaced bands on a gel. The proprietary intelligent SNOW imaging algorithm automatically finds the optimal signal-to-noise ratio (S/N) with minimal input or guesswork from the user.

CCD image optimization