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LI-COR Now Marketing Souped Up Western Blot Assays For Looking at Kinases In-Cell

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It’s caught somewhere in the middle of high-throughput screening and proteomics, but nevertheless, the Odyssey infrared imaging system from LI-COR Biosciences has found a potentially useful niche: Making the common laboratory technique of Western assays fast enough to keep up with the high-throughput demands of drug discovery.

Earlier this month, LI-COR signed a joint marketing and assay validation agreement with Cell Signaling Technology, a company that specializes in producing antibodies specific for the phosphorylation states of various cellular proteins. Combined with LI-COR’s In-Cell Western assays for the Odyssey and IR fluorescent dyes, the antibodies are expected to enable quantitative assays for kinase activation and cell-signaling pathways, the companies said in an official statement.

By introducing this application for the In-Cell Western Assay , LI-COR is entering into the increasingly competitive market for live cell-based kinase phosphorylation assays.

The In-Cell Western Assay, which LI-COR first introduced 14 months ago, is designed to streamline the traditional Western blot assay. In a traditional Western blot assay, cells are lysed; then the contents are loaded onto a gel, blotted, incubated with antibodies, washed, and imaged. It’s worked for many years, but is low throughput and can be error-prone due to the involved protocol.

In an In-Cell Western, however, the lysate preparation and gel loading steps are skipped, according to LI-COR. Instead, permeabilized, fixed cells are labeled in well plates with appropriate fluorescently labeled antibodies. The resulting fluorescence can be read on the company’s Odyssey, which is about the same size as most benchtop gel readers, and is equipped to read 96- or 384-well microplates.

Other assays exist for analyzing proteins in fixed cells, but according to Harry Osterman, LI-COR’s director of technology development, the near-IR and IR capabilities of the Odyssey set the LI-COR assay apart.

“We don’t have background problems — fluorescence caused by cellular materials, the plate, and things like that,” Osterman said. “This allows us to get very good sensitivity inside the cell. There are other systems out there that do chemiluminescent assays inside the cell, but there are various problems with that in terms of it being a dynamic assay — the light generation is continually moving and changing, and it also has background problems.”

Osterman added that the company will be publishing a report soon that compares a chemiluminescent well-based ELISA-type assay against the In-Cell assay, and demonstrates a superior signal and better quanti-fication using the latter. (He did not disclose the publication that would carry this report.)

The instrument was originally developed along with LI-COR’s IRDye 800CW label to improve sensitivity and quantification capabilities for scientists running traditional Western blots. The current Odyssey features two diode lasers — one at 680 nm and one at 780 nm — which allow dual fluorescence imaging, thereby increasing the throughput of the In-Cell Western even more. Along with the LI-COR IRDye for the 780-nm channel, Osterman said the company utilizes a few different Molecular Probes fluorescent dyes, depending on the application, for the 680-nm channel.

Osterman also pointed out that the Odyssey assay is optimized for drug discovery experiments. This is because the background fluorecence produced by the drug candidates is typically out of the IR and near-IR range.

Activated Proteins

The deal with Cell Signaling, which was announced on June 1, enables both companies to offer very specific assays that determine the amount of phosphorylated kinase protein in a given sample. Such assays are crucial in drug discovery programs for indications such as oncology and inflammation; as well as metabolic, cardiovascular, and neurological disease.

“This is primarily a marketing agreement to demonstrate cellular assays that can be performed on LI-COR’s instruments,” said Christopher Bunker, director of business development at Cell Signaling. “Our antibodies are focused on the activation states of signaling molecules like the phosphorylation of kinases.”

Bunker added that there are approximately 518 kinases in the human genome, each of which has one or more roles in common disease states. In addition, he said, common drug targets such as G-protein coupled receptors and ion channels often impact a subsequent kinase pathway, making assays for kinases.

“One of the things you have to do before you do In-Cell Westerns is to make sure your antibodies are very specific, because it will pick up everything,” Osterman said. “With CST we’re going to optimize antibody sets that are very specific for In-Cell.”

“We’re excited about the deal with CST,” added Jim Wiley, marketing manager at LI-COR, “because in terms of a cellular assay, they’re a leader in phosphor-antibodies. And this is a complicated assay, so we’re hoping to make this a lot more straightforward for our customers.”

Osterman admitted that there are microscopy-based platforms on the market that can look at multiple fluorescent dyes in a fixed-cell assay, and that most of these are optimized for dyes in the lower wavelengths.

“If you tried to run an assay at lower wavelengths with [the Odyssey], you’d get much more of a background signal,” he said.

In addition, Osterman said that the In-Cell compares favorably against live-cell assays to emerge recently for drug screening, but costs less. The assay system is currently priced at under $50,000, he said.

“We’re not that far off from having a live cell,” Osterman said. “Basically what we’re doing is freezing it in time with the permeabilization. So we’re not disrupting the inner part of the cell, we’re just breaking open the membrane.” Osterman also said that the company has some customers performing the In-Cell Western assay on fixed tissue sections, but that “we have a problem with resolution with live cells.”

“The actual treatment with a chemical compound is done to the live cell, and then we just freeze it,” Wiley added. “I think most of the experts that we talk to are comfortable with that.” He added that high-content screening with live cells is looking at the exact same thing as the In-Cell Western, but that because things are constantly changing, it requires a very dynamic imaging platform.

LI-COR said that it has about 500 Odyssey instruments installed at various companies and institutions. Wiley said that when the company originally launched the instrument, it was intended for people running traditional Western blot assays, and that this customer base was primarily academic.

The In-Cell Western has a much larger pharmaceutical customer base — Wiley estimates about 80 percent pharma and 20 percent academic. LI-COR said it will continue to focus on broadening and validating a range of assays for the In-Cell platform.

— BB