Capture Your Western Results with These Documentation Systems

You’ve run your sample out on a gel and Coomassie-stained it to make sure it ran properly. You’ve transferred the proteins to a membrane and immunologically probed it to tell you what those bands really are. Now what?

Western blot documentation and analysis have come a long way since radio-tagged antibodies were used to expose X-ray film, a darkroom with toxic chemical baths was needed to develop the film and a protein’s molecular weight and relative quantity were guesstimated by eye. Oftentimes multiple exposures were necessary, because film’s limited dynamic range didn’t allow all the bands to be simultaneously picked up without saturating the produced signal.

Although film is still used by a significant number of researchers, chemiluminescence has largely replaced radioactivity to expose the film.

The bands on the exposed film are often quantitated using a densitometer. 

And film itself is giving way to the digital darkroom, in which chemiluminescent and fluorescent blots are instead imaged by benchtop documentation systems —typically powered by a digital scanner or cooled CCD camera—which record the location, intensity and in some cases the color of the signal emanating from the blot. Here we look at how such instruments help provide an easier, more reproducible and more quantitative workflow for capturing, analyzing and archiving Western blot results.

What are you looking at?

In transitioning to digital detection, researchers have often felt a need to comprise on sensitivity to gain the convenience of the technology. But “the crispness, resolution, etc., have exponentially improved over the years. Now we are able to prove to researchers that we can give them the same sensitivity that they can get with film,” says Taylor Murphy, manager of global applications at Azure Biosystems. Using digital systems, chemiluminescence is generally found to be more sensitive than fluorescence, he notes, and fluorescence in the near-infrared (NIR) range generally outperforms visible-spectrum fluorescence Westerns. Meanwhile, fluorescent blots can be multiplexed. Such insights are crucial to keep in mind when evaluating Western blot documentation systems for your lab’s needs.

Many vendors now offer instruments capable of documenting a variety of chemistries (and a variety of formats, such as PAGE and DNA gels, in addition to Western blots), with user interfaces designed to streamline the process.

For example, the Image Lab Touch touch-screen controlled software on Bio-Rad’s ChemiDoc MP system can “just ask [you] to tell it what fluors you’re using—or are you using chemi? Then based on your selection, the software will pick for you the optimal light source and filter,” explains Raymond Miller, global product manager responsible for the company’s Western blotting product line. “You can also pinch in and out with your fingers, and the system will zoom and be on the right focal plane.”

These systems enable automated or manual lane detection, band detection and molecular-weight calculation. With the appropriate controls, they can calculate signal intensity and thus quantity, and then normalize for loading error. They can also generate a ratiometric value of two distinct proteins or of total protein vs. its phosphorylated form, example.

A simple finger tap

There are a variety of ways to interact with a Western documentation system. For example, ProteinSimple’s FluorChem E, M and R “have a built-in touch-screen interface and computer, with onboard storage. The software is very simple, and the user can do some simple analysis on there,” points out imaging product manager Bonnie Edwards. “Customers can access [images] remotely, if they put them on a network—they can actually pull their images from their iPhone or desk, and they can even control the system from there. It’s basically a web-based interface, so there’s no software they have to install on their phone or computer.”

ProteinSimple also offers three more traditional systems with slightly larger cabinets that require a separate computer running free AlphaView software. AlphaView also can be used for more in-depth analysis of images generated on the E, M and R instruments.

If the capture software and analysis software are one and the same, this could potentially tie up the instrument for another capture while data is being analyzed. This is especially relevant for instruments with multiple users and for instruments relied upon for other tasks, such as gel documentation. One solution is to separate the capture and analysis functions into different pieces of software. Another solution is to let users transfer their data to another device—with the analysis software resident on the device or connected to a network where the software lives. 

Different vendors offer multiple-user licenses, network licenses and even freely available software.

They also use a variety of transfer protocols, including Wi-Fi, Bluetooth, USB stick, Ethernet and direct connection. 

All the data 

Many vendors’ software (as well as third-party software such as ImageJ) can read and analyze files generated by other instruments, if the files are saved in universal formats such as JPG, TIF, BMP or PNG. Yet there may be information, including metadata and dynamic range, lost in the transfer from the instrument’s proprietary “raw” file format—especially if it’s not exported properly.

It’s generally agreed that the software that comes with the instruments gives users the capabilities they need to analyze their Western blots, and there is no need to look elsewhere. Thus Jeff Harford, senior product marketing manager for LI-COR, suggests “that people use the software that came with their imager … because they know the best about what is in that data.”

An exception may be found withmore specialized applications, though. David Cool, professor of pharmacology and toxicology at Wright State University, bemoans the fact that expensive software is needed to automatically handle two-dimensional gel and blot analysis. His students, he says, instead have to draw circles around the spots using the software that came with the instrument—although he believes that a simple tweak should enable the standard software to handle 2D.

Today’s Western blot documentation systems are able to handle the vast majority of what’s asked of them. Not only can they automatically detect, quantitate and determine the molecular weight of proteins, normalize to standards and allow for a far greater (some claim unsaturable) dynamic range than traditional X-ray film, but they’re often 21st-century-enabled for user-password protection, data security (including CFR 21 Part 11 documentation), remote access and the ability to generate publication-ready images. It’s no wonder so many new labs are opting for a digital darkroom, and established labs are making the switch.

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