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Assay Method Information |
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| | Human KLK7 Fluorescence-Lifetime Assay |
| Description: | Recombinant human KLK7 (5 nM concentration) was pre-incubated with inhibitor at various concentrations for 1 h at room temperature in 50 mM sodium citrate buffer at pH 5.6, containing 150 mM NaCl and 0.05% (w/v) CHAPS. The enzyme reaction was initiated by addition of the substrate Ac-Glu-Phe-Lys-Pro-Ile-Leu-Trp-Arg-Leu-Gly-Cys(PT14)-Glu-NH2 (0.8 μM; Biosyntan, Berlin, Germany, product BS-#7599). Fluorescence-lifetime measurements were conducted on an Ultra Evolution fluorescence lifetime reader (TECAN, M nnedorf, Switzerland). The excitation light source was a semiconductor laser at 405 nm, producing picosecond light pulses with a selected repetition frequency of 10 MHz. The emission was collected through a 450 nm bandpass filter with 25 nm bandwidth. The measurement time per well was set to 1 s, yielding approximately 1,000 counts in the peak channel. The parameters used as assay readout were the mono-exponential lifetimes. |
| Affinity data for this assay | |
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