The Unlikelihood of an “Eureka Moment”

Adriane Thompson’s Mentored Advanced Project (MAP) experience didn’t deter her from a future in research. However, the fourth-year biology major says, “It gave me a really good reality check about how frustrating research can be.”

Thompson ’24 spent the summer in the lab of Benjamin DeRidder, associate professor of biology, working with a prolific protein known as rubisco activase.

“Rubisco activase is one of the most important proteins in plants and one of the most abundant proteins on the planet,” Thompson explains. The protein is necessary for photosynthesis, but it’s also incredibly sensitive to heat.

Amidst ongoing global warming, researchers like DeRidder are studying how plants respond to heat stress and what biological systems might be in place to protect them. Recently, his lab discovered that a version of this rubisco activase protein — the “β2 isoform” — has higher mRNA levels in plants exposed to heat stress. This doesn’t necessarily mean that the plants are producing more β2, but it does mean that they are producing more of the genetic instruction manuals that could be used to make β2.  

With research partner Anastasia Rompi ’25, Thompson set out to explore whether these high mRNA levels correlate with higher levels of the actual β2 protein in heat-stressed plants. “If so, it would seem to indicate that there is something about this isoform that is better for plants to survive heat stress and continue photosynthesis,” she explains.

When Research Outgrows the Technique

The first step in Thompson’s project was to develop a protocol for isolating super pure rubisco activase β1 and β2 proteins from plant tissue. Once she’d isolated the proteins, she had to find a way to measure their relative quantities. This is where things got complicated.

“There’s an issue in that β1 and β2 isoforms are super, super close in size, so we can’t use traditional techniques to differentiate between the two proteins,” Thompson says. Gel electrophoresis, a technique widely used by scientists to separate proteins by size, doesn’t work to separate these two proteins. They both show up as one big clump. Instead, Thompson and her lab partner aimed to compare the relative amounts of the two isoforms using a technique new to the DeRidder lab: mass spectrometry.

The Importance Of Trial And Error

Mass spectrometry is an analytical technique that measures proteins that have been bound by unique, specific antibodies. This binding process is called immunoprecipitation and it’s notoriously finicky. Finding an antibody that will successfully bind to your target protein — and only your target protein — can require a lot of trial and error. This was certainly Thompson’s experience.

Try as they might, Thompson and Rompi just couldn’t bind enough pure rubisco activase protein to analyze with mass spectrometry. “It was unfortunate but pretty realistic,” Thompson says. “We were trying to optimize a protocol, something that can take months and months, and unfortunately we didn’t have the time in order to get the results we wanted.”

Thompson’s understanding of just how long it can take to develop a working protocol has been one of the critical takeaways of her summer: “It’s one of the realities of a lot of research. Optimization is a critical aspect of almost every scientist’s career — but that doesn’t mean that you will have that ‘eureka’ moment, especially not in only 10 weeks,” says Thompson.

The Unexpected Joy Of A Hail Mary

For Thompson, the silver lining of her immunoprecipitation conundrum was a period of creative experimentation few undergraduate researchers get to experience. “Our setbacks gave us the independence to say, ‘Well, if we’re in this position where we don’t have more funds or time to buy new things, we’re just going to try to get as much information as possible.’”

In the last few weeks of the project, Thompson and Rompi paused their work on the immunoprecipitation protocol they’d hoped to perfect and returned to the gel electrophoresis technique they’d ruled out at the start of the summer. “We tried things that felt silly or probably ineffective because we had the ability to, and there’s something really fun about that,” says Thompson.

And with their Hail-Mary experimentation, they finally found some success. By running the gel electrophoresis for much longer than normal, they found that they could begin to differentiate the β1 and β2 proteins. “We got some really interesting results in terms of general information about the products that we had been using this whole time that maybe we wouldn't have understood otherwise.”

The summer involved frustration, false starts, and lots of nitty gritty trial and error. “But at least now I have a little better of a mindset to go tackle that kind of thing with,” Thompson reflects. “Because you just can’t have results be the daily thing that’s keeping you going.”