| Assay Method Information | |
| | Factor XIa Inhibition Assay Utilizing a Fluorophore-Quencher Pair Peptide Substrate |
| Description: | A fluorescence intensity (FLINT) based assay was used to monitor inhibition of Factor XIa. The peptide substrate, 5Fam-KLTRAETV-K5Tamra (purchased from New England Peptide) was chosen based on the FXI sequence. Conversion of zymogen FXI to its activated form, FXIa, occurs by proteolytic cleavage by FXIa at two sites, Arg146 and Arg180. The custom peptide used in this assay was based on the Arg146 cleavage site of FXI. The peptide substrate was designed with a fluorophore-quencher pair, where the fluorescence is quenched until FXIa cleaves the 8-mer peptide after the Arg residue. The substrate KM was fit to a substrate inhibition model whereby kcat=0.86 s−1, KM=12.4 μM, Ki=61.6 μM with an enzymatic efficiency, and kcat/KM=69523 M−1 s−1. The Factor XIa FLINT assay was used with the following 5Fam-KLTRAETV-K5Tamra assay buffer: 50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM CaCl2), 0.1 mg/mL BSA, 0.03% CHAPS. Assay buffer was prepared by mixing all ingredients fresh. 5Fam-KLTRAETV-K5Tamra peptide substrate was first prepared at 10 mM in 100% DMSO, then diluted to 3 mM in 100% DMSO. Assay buffer was then added directly to the 3 mM stock of substrate to prepare the 30 μM 2× working concentration (15 μM final concentration). The 2×Factor XIa stock solution was prepared by diluting 6.562 μM stock in 1×assay buffer for a 200 μM working stock solution (100 μM final concentration). |
| Affinity data for this assay | |
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