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Assay Method Information

Assay Name:  Binding Assay
Description:  Purified, recombinant FGFR4 was pre-incubated with 10 μM compound overnight at 4° C., or for 1 hour at room temperature. Following pre-incubation, protein samples were separated using SDS-PAGE and gels were stained with SimplyBlue™ SafeStain (Life Technologies, Grand Island, N.Y.). FGFR bands were cut out and digested using an In-Gel Tryptic Digestion Kit (Thermo Scientific, Waltham, Mass.). Digested samples were run on a Thermo Scientific Q Exactive™ LCMS using reverse phase separation and tandem mass spectrometry to identify modified peptides.Alternatively, following pre-incubation FGFR4 was concentrated and buffer exchanged on an OPTI-TRAP protein concentrating and desalting C4 column (Optimize Technologies). Protein was eluted in acetonitrile containing 0.1% formic acid and run by direct injection on a Thermo Scientific Q Exactive™ LCMS to identify modified, intact FGFR4.
Affinity data for this assay
 

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Last update November 1, 2007
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