Babco, Signet, Sternberger Monoclonals, Senetek, Covance

protein

Recombinant Human KLK3 (carrier-free)

761406

100 μg

698 USD

BA

761406

Recombinant Human KLK3 (carrier-free)

Prostate-specific Antigen, PSA, Kallikrein 3

100 μg

698 USD

Human

ADD EXPRESSION SYSTEM HERE

RECOM

Human KLK-3 Activity Assay with fluorogenic substrate;Human KLK-3 activity is measured by its ability to cleave a fluorogenic peptide substrate Suc-Arg-Pro-Tyr-AMC after hKLK-3 is activated by thermolysin. The progress of the substrate cleavage is monitored by increase in intensity of fluorescence emission at 460 nm with excitation at 380 nm.Materials and Buffers;Activation Buffer: pH 7.5, 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35.;Assay Buffer: pH 8.0, 50 mM Tris, 1.0 M NaCl.;500 mM EDTA solution at pH 8.0 for stopping the activation.;Bacterial Thermolysin (TLN) (SIGMA, Cat # P1512).;Human KLK-3: BioLegend;KLK-3 substrate: Suc-Arg-Pro-Tyr-AMC;Assay Procedures;Dilute TLN to 0.5 µg/mL in Activation Buffer.;Dilute hKLK-3 to 200 µg/mL in Activation Buffer.;Combine equal volumes of 200 µg/mL of hKLK-3 and 0.5 µg/mL TLN to make the activating concentration of 100 µg/mL of hKLK7 and 0.25 µg/mL of TLN.;Incubate at room temperature for 1 hour.;Stop the reaction with equal volume of 500 mM EDTA at pH 8.0. At this moment, hKLK-3 is at 50 µg/mL.;Dilute the activated hKLK-3 to 4 µg/mL with Assay Buffer.;Dilute the substrate stock at 2 mM in Assay Buffer.;Load 50 µL of the activated hKLK-3 in 4 µg/mL into a plate, and start a reaction by adding 50 µL of 2 mM of the substrate solution into each well.;Read the reaction progress by monitoring fluorescence emission at 460 nm with excitation at 380 nm in kinetic mode for 5 minutes.;The final human KLK-3 concentration is at 2 µg/mL (0.2 µg), the substrate concentration at 1 mM, TLN at 0.005 µg/mL, and EDTA at 10 mM.;Conversion factor of AMC is 0.3 pmol/RFUHuman KLK-3 Activity Assay with chromogenic substrate;Human KLK-3 activity is measured by its ability to cleave a fluorogenic peptide substrate Suc-Arg-Pro-Tyr-pNA after hKLK-3 is activated by thermolysin. The progress of the substrate cleavage is monitored by increase in intensity of absorbance at 405 nm.Materials and Buffers;Activation Buffer: pH 7.5, 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35.;Assay Buffer: pH 8.0, 50 mM Tris, 1.0 M NaCl.;500 mM EDTA solution at pH 8.0 for stopping the activation.;Bacterial Thermolysin (TLN) (SIGMA, Cat # P1512).;Human KLK-3: BioLegend;KLK-3 substrate: Suc-Arg-Pro-Tyr-pNA;Assay Procedures;Dilute the TLN to 0.5 µg/mL in Activation Buffer.;Combine equal volumes of provided 200 µg/mL of hKLK-3 and 0.5 µg/mL TLN to make activating concentration of 100 µg/mL of hKLK7 and 0.25 µg/mL of TLN.;Incubate at room temperature for 1 hour.;Stop the reaction with equal volume of 500 mM EDTA at pH 8.0. At this moment, hKLK-3 is at 50 µg/mL.;Dilute the activated hKLK-3 to 10 µg/mL with Assay Buffer.;Dilute the substrate stock at 2 mM in Assay Buffer.;Load 50 µL of the activated hKLK-3 in 10 µg/mL into a plate, and start a reaction by adding 50 µL of 2 mM of the substrate solution into each well.;Read the reaction progress by monitoring absorbance at 405 nm in kinetic mode for 5 minutes.;The final human KLK-3 concentration is at 5 µg/mL (0.5 µg), the substrate concentration at 1 mM, TLN at 0.0125 µg/mL (1.25 ng), and EDTA at 25 mM.;Conversion factor of pNA at pH 8.0 by using extinction coefficient 8800 M^-1cm^-1, 0.32 cm of light path correction, and total volume of 100 µL: 35.5 pmol/mAu.This protein is in the latent form and needs to be activated for bioassay.;BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue-ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are verified in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at tech@biolegend.com.;