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May 28, 2020

Save time in Western blotting

By Cytiva

How much time do you spend finding the right exposure settings for Western blots? Find out how the right CCD imager can help you work more efficiently, freeing valuable resources to drive your research forward, faster.

Western blotting efficiency in the lab

Using time in the lab efficiently promotes success in research with faster generation of key data leading to scientific breakthroughs. To drive efficiency, it is important to watch for ways to improve and refine routine practices and procedures, as time savings in these daily repetitive activities can add up quickly.

Western blotting is an affordable process that reliably answers important basic questions regarding proteins of interest. For this reason, Western blots, or protein immunoblots, are used daily by most labs working on protein-based research.

While the Western blotting method has stayed largely unchanged over the last few decades, there remains opportunity to increase efficiency in the process. Saving even just a little bit of time on every Western blot can add up to substantial time-savings over the course of a week, month, or year—time you and your colleagues can spend on other valuable research efforts.

Overview of the dry powder milling process at all three manufacturing sites.

Optimizing exposure times in CCD imaging

Western blots often have bands of varying intensity, with low-expression proteins presenting with lower band intensity and highly expressed proteins represented by more intense bands. Low-intensity bands need longer exposure times to be visible, while the high-expression bands require shorter exposure times to avoid saturation.

The varying imaging requirements of low- and high-expression proteins make visualizing both on the same blot a challenge. Although the introduction of digital imagers has provided substantial improvements over X-ray films, the dynamic range of many CCD imagers today remains limited. The user has to choose the optimal exposure settings by trial-and-error, and it is often not possible to visualize both low- and high-intensity bands on the same blot without some trade-off.

Another challenge of this trial-and-error approach is the race against time to find the optimal exposure conditions, usually requiring multiple test exposures to get the best possible image. As the chemiluminescent reaction progresses, the signal-to-noise ratio (SNR) peaks and begins to drop until the signal is indistinguishable from noise. There is also a risk that membranes dry out over time.

Once the chemiluminescent reaction progresses and the signal becomes weaker or undetectable, it might be necessary to redo the blot if you have not yet been able to find optimal exposure conditions. If you do have an image, the SNR might limit your ability to quantitate across strong and weak bands simultaneously.

Some CCD imagers offer a function that adjusts exposure time automatically; however, this function is based on the brightest signal on the blot and cannot improve the SNR of the images. This approach compromises the visibility of lower intensity bands, which are possibly the bands you are most interested in seeing.

Find out more about the importance of SNR

SNOW mode saves time with automatic signal-to-noise optimization

The key to saving precious time when using Western blots for protein analysis is capturing an image with optimal SNR quickly and reliably. Our proprietary intelligent algorithm, the SNOW (signal-to-noise optimization watch) imaging mode, can help you detect weak bands without saturating strong bands and achieve high sensitivity without compromising image quality.

SNOW imaging is available with the Amersham ImageQuant 800 biomolecular imager (Fig 1). When using this imaging mode, the system continually checks and averages image intensity data from multiple exposures to automatically optimize the Western blot image. The imaging algorithm stops the process once the SNR peaks to acquire the best possible image. This method eliminates the need to spend time optimizing exposure and capture settings and stretches the dynamic range without compromising sensitivity and resolution.

The ImageQuant 800 biomolecular imager helps save time in Western blot imaging

Fig 1. The Amersham ImageQuant 800 biomolecular imager helps save time in Western blot imaging.

If you need to quantitatively analyze both high signal and weak bands, trust the SNOW imaging mode to automatically optimize exposure time and maximize SNR; the output is a single image file, ready for analysis.

Remote Image transfer and scheduling

Easy-to-use software on the Amersham ImageQuant 800 system can help improve research efficiency by enabling remote system management, saving you a trip to the lab. The ImageQuant CONNECT software allows you to schedule equipment usage and access image files from your desk (Figs 2, 3, and 4).

The ImageQuant CONNECT software allows for remote access to imaging and scheduling.

Fig 2. Use ImageQuant CONNECT software from your office to access images and the scheduler tool on board the instrument.

The ImageQuant CONNECT interface can be used to view the status of all the Amersham ImageQuant 800 instruments connected to the same local network in your facility.

Fig 3. The ImageQuant CONNECT tool can be used to view the status of all the Amersham ImageQuant 800 instruments connected to the same local network in your facility. This capability allows you to choose an available instrument and plan your experiments.

Perfect for a busy multi-user lab environment, the on-board scheduler application can be used to block time on the instrument to plan and run your experiment.

Fig 4. Perfect for a busy multi-user lab environment, the on-board scheduler application can be used to block time on the instrument to plan and run your experiment. Easily access the scheduler from your office via the remote ImageQuant CONNECT software to view and manage bookings.

The Amersham ImageQuant 800 control software includes automatic color marker overlay, saving time by automatically imaging and overlaying white light color marker images with chemiluminescence images of the same blot. This approach makes it easier to determine the molecular weight of your bands of interest. With most other imagers, users have to take separate white light and chemiluminescent images and manually overlay them in a separate analysis software. The Amersham ImageQuant 800 also automatically provides users with both grayscale and colored images.

High quality, efficient CCD imaging

Small time savings in tasks that are part or many researchers’ daily routine lead to improvements in overall research productivity over time.

Designed with features such as SNOW imaging and ImageQuant CONNECT software, the Amersham ImageQuant 800 system generates the best possible images with improved data handling to deliver the ultimate solution for fast, efficient Western blotting.

We provide a range of solutions for optimizing Western blot imaging and workflows. To develop a deeper understanding of the methods and principles behind CCD imaging, access our imaging principles and methods guide. For further support and inquiries relating to any aspect of the Western blotting or the CCD imaging workflow, please contact our Scientific Support team.

Find out how the ImageQuant 800 system can support your Western blotting workflow


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