Babco, Signet, Sternberger Monoclonals, Senetek, Covance

protein

Recombinant Human TIMP-4 (carrier-free)

767504

25 μg

440 USD

BA

767504

Recombinant Human TIMP-4 (carrier-free)

Tissue inhibitor of metalloproteinases 4, TIMP-4, TIMP4

25 μg

440 USD

Human

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RECOM

Human TIMP-4 Activity assay; Human TIMP-4 activity is measured by its ability to inhibit cleavage of a fluorogenic peptide substrate Mca-PLGL(Dpa)AR by activated hMMP-2. The potency of hTIMP-4 is described as IC50 value.; Materials;Assay Buffer: TCNB (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 7.5);Recombinant Human MMP-2 (Predicted MW: 73.2 kDa);Recombinant human TIMP-4 (Predicted MW: 23.9 kDa);MMP-2 fluorogenic substrate: Mca-PLGL(Dpa)AR;P-Aminophenylmercuric acetate (APMA); Activity assay procedures;Dilute hMMP-2 in the Assay buffer at 100 µg/mL.;Activate hMMP-2 by adding APMA to a final concentration of 1 mM.;Incubate the activating mixture for 1 hours at 37 °C.;Dilute the activated hMMP-2 to 4 ug/mL (0.4 ng/µL) in Assay Buffer.;Make serial dilution of hTIMP-4 in Assay Buffer from 16000 ng/mL to 7.81 ng/mL, and include 0 ng/mL as control.;Add into a tube 25 µL of each serially-diluted hTIMP-4 including the control, and then add 25 µL of 4 µg/mL activated hMMP-2.;Incubate the mixture at 37 °C for 2 hours.;Add 200 µL of Assay Buffer into each tube prepared in step 6 to make an assay mixture.;Dilute the substrate in Assay Buffer at 20 µM.;Load into a well plate 50 μL of each assay mixture and start the reaction by adding 50 μL of 20 μM Substrate. Include a substrate blank containing 50 μL Assay Buffer and 50 μL of 20 μM Substrate.;Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.;Analyze the data with non-linear regression to estimate IC50 value of hTIMP-4 towards hMMP-2.; Final assay conditions per well;hMMP-2: 20 ng (200 ng/mL);hTIMP-2: 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.57, 0.781, 0 ng/mL;Substrate: 10 µM;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.;