Babco, Signet, Sternberger Monoclonals, Senetek, Covance

protein

Recombinant Human Cathepsin L (carrier-free)

787506

100 μg

932 USD

BA

787506

Recombinant Human Cathepsin L (carrier-free)

CTSL, Major Excreted Protein (MEP), CATL

100 μg

932 USD

Human

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RECOM

Human Cathepsin L Assay Procedure Human Cathepsin L activity is measured by its ability to cleave the fluorogenic peptide substrate Z-LR-AMC. The accumulation of cleavage product is quantified by the increase in fluorescence at 460 nm upon excitation at 380 nm. Materials and Buffers 1. ;Assay Buffer: 50 mM MES, 5 mM DTT, 1 mM EDTA, 0.005% (w/v) Brij-35, pH 6.0;2. ;Human CTSL substrate: Z-LR-AMC (Bachem Cat. No. I-1960);3. ;Recombinant human cathepsin L;4. ;F16 Black Maxisorp Plate (Nunc, Cat. No. 475515);5. ;Fluorescent Plate Reader (Model: SpectraMax Molecular Devices) or equivalent Assay Procedure 1. ;Dilute the human CTSL to 40 µg/mL in assay buffer.;2. ;Incubate the diluted human CTSL on ice for 15 minutes.;3. ;Dilute the human CTSL to 0.08 ng/µL in assay buffer. Make six 1:2 dilutions in a black well plate. Include a blank with 50 µL assay buffer.;4. ;Dilute the substrate to 80 µM in assay buffer.;5. ;Start the reaction by adding 50 µL of the diluted substrate to each well. 6. ;Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.;7. ;Calculate specific activity: Specific Activity (pmol/min/µg) = Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU) amount of enzyme (µg) ; *Adjusted for Substrate Blank **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891). Per well:;• ;Human CTSL: 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 ng;• ;Substrate: 40 µM ;