InnoZymeTM Cathepsin B Activity Assay Kit; Fluorogenic

InnoZymeTM Cathepsin B Activity Assay Kit; Fluorogenic

Stock Code: 3586712
Manufacturer Part No: CBA001-1KIT
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Analysis Note


Positive ControlCathepsin B


The data obtained can be displayed in two ways:1. As fluorescence units: correct the fluorescence value of all samples by subtracting the value of the blank, and then calculate the mean fluorescence value for each sample in duplicate. For the biological samples also subtract the value of the sample assayed with inhibitor.OR2. As µmole of free AMC/mg total protein/time (min.): calculate the mean fluorescence value as stated above. Plot a graph correlating the mean fluorescence values of the AMC standards (y-axis) to their concentration in µmole (x-axis). The cathepsin B activity of the unknown samples can be interpolated from the standard curve.Calculate the amount (µmol) of free AMC per mg of total protein and time unit.


Components


Cathepsin B Enzyme, Calibration Standard, Cathepsin B Substrate, Assay Buffer, Cathepsin B Inhibitor, Reduction Reagent, Cell Lysis Buffer, Microtiter Plate, Plate Sealer, and a user protocol.


General description


A sensitive fluorogenic assay for the measurement of cathepsin B activity (Excitation max: ~360-380 nm; Emission max: ~430-460 nm). Cathepsin B is a cysteine proteinase which has been associated with increased invasivessness and development of the malignant cell phenotype. Cathepsin B has also been implicated in inflammatory airway disease.


Legal Information


CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany


Other Notes


Bervar, A., et al. 2003. Biol. Chem. 384, 447.Grigolo, B. et al. 2003. Biomaterials 24, 1751.Scolaris, A., et al. 2002. Biol. Chem. 383, 1297.Murata, M., et al. 1991. FEBS Lett. 280, 307.Berquin, I.M. and Sloane, B.F. 1996. Adv. Exp. Med. Biol. 389, 281.Burnett, D., 1995. Arch. Biochem. Biophys. 317, 305.Reddy, V.Y., et al. 1995. Proc. Natl. Acad. Sci. 92, 3849.Buttle, D.J., 1994. In: Immunopharmacology of Joints and Connective Tissue (Dingle, J.T. and Davies, M.E., eds) London: Academic Press. 225.Barrett, A.J. and Kirschke, H., 1981. Methods Enzymol. 80, 535.


Packaging


1 kit in Glass bottle


Preparation Note


Dilute samples with dilution buffer as necessary. Note: if the measured FU exceeds 9,000 the sample should be diluted further. Cell lysate: wash cell pellet with ice-cold PBS. Add 500-1000 µl of cell lysis buffer (~1 ml per 1x107 cells) and incubate on ice for 30 min. Vortex and centrifuge the lysate at 14,000 x g in a pre-cooled tabletop microcentrifuge. Immediately transfer the supernatant to a fresh microcentrifuge tube and discard the pellet. Dilute the lysate 1:5 or 1:10 before determining the protein concentration via BCA protein assay.


Dilution Buffer: dilute assay buffer 1:10 with dH2OExample: add 0.5 ml of assay buffer to 4.5 ml of distilled water. Standard: the free AMC standard is supplied as a 2 mM stock (100-fold concentrated) solution. To create a calibration curve prepare serial dilutions of the standard ranging from a concentration of 20 µM to a concentration of 1

Quality Level100
ManufacturerSIGMA-ALDRICH

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