The total FGFR2 kit monitors the cellular FGFR2 expression level and can be used as a normalization assay for the phospho-FGFR kit.
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  • Ease-of-use
  • Highly specific
The total FGFR2 kit monitors the cellular FGFR2 expression level and can be used as a normalization assay for the phospho-FGFR kit.
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Overview

The Total FGFR2 cellular assay monitors total FGFR and is used as a normalization assay with the phospho-FGFR kit. Mutations in FGFR2 have been associated with cancer (breast, melanoma) and abnormal bone development (cranosynostosis syndromes).

Total-FGFR2 assay principle

The Total-FGFR2 assay quantifies the expression level of FGFR2 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-FGFR2 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of FGFR2 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Total-FGFR2 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total FGFR2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-FGFR2 1-plate assay protocol

Detection of total FGFR2 with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Detection phospho and total FGFR2 on Kato-III cells

Kato-III cells were plated in 96-well plate and incubated overnight. Cells were treated with increasing concentration of FIIN-1 hydrochloride compound for 6h at 37 °C - 5% CO2. After stimulation, cells were washed and lysed with lysis buffer#2 1X for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho FGFR2 or total FGFR2 detection reagents were added. The HTRF signal was recorded after a overnight incubation at room temperature.
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