Modernizing Western Blotting: A Digital Solution for Protein Analysis

The early days of digital western blot imagers were rough. Like the first digital cameras, the best photographs they produced were blunt and grainy, orders of magnitude below old and reliable film’s quality. A few decades ago, professional photographers wouldn’t be caught dead with digital cameras – until the modern DSLR proved it could be just as sharp and sensitive and malleable as the most skilled darkroom technician. Today, that digital revolution seems to have finally hit western blotting.

Digital western blot imagers today now rival – and surpass – film (see Table at bottom).

Yet, x-ray remains king in protein detection because it readily picks up signals from the blot.

But many researchers know this supposed strength can actually be a pitfall. Without the right kind of darkroom skills and experience, many scientists accidentally oversaturate their signal through overexposure or use the wrong settings for particular target proteins.

This article reports on three researchers at different labs who have traditionally used x-ray film to develop western blot images and their experience testing it side-by-side with digital imaging. What they’ve found is that digital imaging is more sensitive, straightforward, and flexible than using film x-ray films.

Digital imaging resolves minute differences in ubiquitinated proteins

It’s a problem that Ling Shao, an assistant professor of medicine at the University of Southern California, knows well. He studies proteins that play a role in programmed cell death in cancer. One protein he studies closely is called RIPK1, which scientists think is a critical signal in a cell’s self-destruction. Cells regulate these proteins through modifications called ubiquitination, but those changes are minute and the altered proteins are similar enough that sensing them is a careful and laborious process.

In a western blot, RIPK1 proteins appear as thinly spaced bands. If the exposure is too long using film, the signal oversaturates and drowns itself out. “On film, it’s quite easy to overexpose the blot, leading to bands that fuse together,” Shao said. “Then you might have to re-expose several times to get a usable image, if it’s even possible at all.”

He doesn’t have to deal with that using digital imaging (ChemiDoc Touch from Bio-Rad Laboratories). He says this digital imager has high enough resolution that even bright signals from different proteins developed with strong substrates will be distinct. “Because the imager can capture all these shades of gray, you can delineate all the different bands where, on film, it just appears as a single band,” he said.

This same sensitivity saves him time. “I don’t have to continually try to determine the optimal substrate for the membrane,” Shao said. “You just put on a strong substrate,” and the machine does the rest. Shao said that’s all it takes to get the resolution he needs.

Digital imaging’s wider dynamic range enables researchers to study low and high expressing proteins simultaneously

Modern digital imaging technology doesn’t just have more resolution than traditional film imaging, it has a wide dynamic range for light detection. Aiwen Dong, a post-doctoral fellow in gastroenterology and hepatology at Stanford University, said that advantage has saved his research in the past.

He studies skin growth proteins in an effort to realize why certain cancers like colorectal cancer forms in the gut. But those proteins are hard to detect because cells make them at very low levels, and they need long exposure times in order for anything to show up on film. When their images do materialize, they appear as feeble, faint bands. But not all skin growth proteins are made equally – some are more plentiful than others and have a stronger signal.

That becomes a problem in trying to compare different proteins at the same time. “I want to compare the intensities of different bands,” Dong said. But in trying to catch a glimpse of those faint bands, the more plentiful proteins overwhelm the signal when Dong exposes the film long enough for the dimmer bands to show up. “But with the ChemiDoc Touch, you can still do quantification between bands,” he said. “The ChemiDoc Touch gives you a box to select your area of interest, so you can let the machine decide the best exposure time to get the best dynamic range.”

Transforming the work environment: fast, reliable, immediate, and accessible

Ernesto Diaz-Flores, an assistant professor of medicine at the University of California, San Francisco, often has the same problems that Dong has. He also needs to compare many different proteins at once. For him, the goal is to understand proteins that regulate survival pathways for childhood leukemia cells. He uses an approach that measures 25 to 30 different proteins simultaneously, which might all have varying concentrations in the cell.

“Having a very strong band next to a very faint band that posed a special challenge,” he said. “Using film, you had to first try to expose for the dark band then the faint band, and it was always too much exposure or not enough.” He found himself getting frustrated with the wasted time over film’s technical limitations.

“What really made me switch from film to digital imaging was the speed,” Diaz-Flores said. “I didn’t have to guess anymore for how long I was going to expose the film and then wait to see the results and then try again. I just hit the button and have the results right away.”

Digital imaging has really transformed his work environment. “You don’t need the darkroom anymore. You’re in a light and bright room in a small space with a small machine,” Diaz-Flores said. It’s a new era, he repeated. “Now we are able to change or crop [the image], so the bands you want to be looking at aren’t in the background,” he said. “You can send your pictures straight to your computer. You can get it in a USB drive. So, it’s a very fast, very reliable way to get your protein analysis data as opposed to the old way of conventional film approach.”

He said that’s the best thing about going digital. Everything is more immediate, more accessible, and more efficient.