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Invitrogen’s TR-FRET CBA Uses Terbium Donor, Interrogates JAKs and JAK/STAT

BOSTON — Invitrogen has developed a suite of biochemical and cellular assay tools for interrogating the JAK/STAT pathway and identifying inhibitors of the JAK family of protein kinases, a development the company believes can be a high-throughput alternative to ELISAs.
The company used a time-resolved FRET cell-based assay approach to determine the phosphorylation state of the STAT3 protein as a readout of JAK activity, Tina Hallis, a research area manager at Invitrogen, told CBA News at the Assay Development and HTS Screening conference, held here last week.
In a follow-up interview with CBA News this week, Hallis said that Invitrogen’s TR-FRET technology uses terbium as the TR-FRET donor, whereas other sources commonly use europium.
She explained that unlike europium, terbium can be paired with red and green fluorophores, allowing more physiologically relevant assays to be developed by expressing GFP fusions of native protein substrates in the very cells that are being assayed.
In Invitrogen’s LanthaScreen STAT3 cell lysate assay, the investigators used a GFP-STAT3 fusion as the substrate for JAKs, which allows for direct TR-FRET between the terbium-labeled anti-pY705 STAT3 antibody and the GFP, said Hallis.
“Similar approaches from other sources are very clunky because additional components must be added to the assay in order to bring the donor and acceptor pair together,” Hallis said.
She said that the Invitrogen system uses a genetically encoded GFP acceptor as part of the substrate, which simplifies the design of this TR-FRET cellular assay.
To complement the target-specific cell lysate assay, the researchers have also designed cell-based assays, called CellSensor, to generally interrogate JAK/STAT pathways using a beta-lactamase reporter gene downstream of relevant response elements for various STATs.
The CellSensor is commercially available and Hallis said that the company is continuing to add disease-relevant cell backgrounds.
Hallis said Invitrogen is offering the LanthaScreen TR-FRET cell-based assay for STAT3 and STAT1 phosphorylation as custom cell lines, though they will be launched as catalog items later this year. Invitrogen plans to expand its cell lysate assays to cover all of the STATs, said Hallis.

“The Invitrogen system uses a genetically-encoded GFP acceptor as part of the substrate, which simplifies the design of this TR-FRET cellular assay.”

Inhibitors of the JAK family of kinases are being sought to treat various malignancies and immune disorders. “Researchers are finding that mutations [in these genes] can cause constitutive activity in these signaling pathways,” said Hallis. She explained that these mutations and anomalous expression of JAK protein result in mis-regulation of these pathways, causing aberrant control of genes, such as those involved in cell differentiation.
This aberrant control comes into play in conditions such as myeloid proliferative disorders, in which a consistent mutation in the JAK2 protein has been discovered, Hallis said.
Hallis said the company recognizes that the JAK and JAK/STAT pathways are very important, so it has placed considerable effort into developing biochemical and cell-based tools to study them.
“In the purified kinase area, we have all of the four JAKs and the disease-relevant JAK2 mutant, so we have a biochemical way to study this disease-relevant JAK2 mutant, and we also have a cell-based reporter assay to study that same mutant,” she said.

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