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The Cytochrome c Oxidase Assay Kit

The Cytochrome c Oxidase Assay Kit
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Cat. #: CB008

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The Cytochrome c Oxidase Assay Kit 

Cat. #: CB008

Size: 100 Tests

Description

The Cytochrome c Oxidase Assay Kit is designed for the determination of cytochrome c oxidase activity in soluble and membrane bound mitochondrial samples. Cytochrome c oxidase is the last enzyme in the respiratory electron transport chain of mitochondria. Its main function is to convert molecular oxygen to water and aid in establishing mitochondrial membrane potential. Cytochrome c oxidase locates to the inner membrane which separates the mitochondrial matrix from the intermembrane space. This colorimetric assay is based on observation of the decrease in absorbance at 550 nm of ferrocytochrome c caused by its oxidation to ferricytochrome c by cytochrome c oxidase. This kit is suitable for detection of mitochondrial outer membrane integrity and for detection of mitochondria in subcellular fractions.

Components

This kit is sufficient for 100 tests.

Assay Buffer:  25 ml, 50 mM Tris-HCl, pH 7.0, containing 600 mM KCl.

Enzyme Dilution Buffer:  20 ml, 20 mM Tris-HCl, pH 7.0, containing 500 mM sucrose

Cytochrome c: 50 mg

1 M Dithiothreitol (DTT): Solution 0.4 ml, 1 M DTT in deionized water

Cytochrome c Oxidase(positive control):  1 vial

n-Dodecyl b-D-maltoside: 10 mg

1X Assay Buffer: 10 mM Tris-HCl, pH 7.0, containing 120 mM KCl – Dilute an aliquot of Assay Buffer 5-fold with water. Keep at room temperature (~25 °C).

1X Enzyme Dilution Buffer: 10 mM Tris-HCl, pH 7.0, containing 250 mM sucrose – Dilute an aliquot of

Enzyme Dilution Buffer 2´ (E2903) 2-fold with water. Keep at 2–8 °C.

Enzyme Dilution Buffer with 1 mM n-Dodecyl β-D-maltoside (for measurement of mitochondrial

integrity): 10 mM Tris-HCl, pH 7.0, containing 250 mM sucrose and 1 mM n-dodecyl-b-D-maltoside –

Dissolve 1.02 mg of n-Dodecyl-b-D-maltoside (D4641; MW 510.6 Da) in 2 ml of 1´ Enzyme Dilution Buffer.

0.1 M Dithiothreitol (DTT) Solution: Dilute an aliquot of the 1 M DTT Solution (D7059) 10-fold with ultrapure water to a concentration of 0.1 M.

Ferrocytochrome c Substrate Solution (0.22 mM): Dissolve 2.7 mg of cytochrome c (MW 12,384 Da) in

1 ml of water. In order to reduce the protein, add 5 ml of the 0.1 M DTT Solution to a final concentration of

0.5 mM, mix gently, and wait for 15 minutes. The color of the solution should go from dark orange-red to pale purple-red. Measure the A550/A565 ratio of an aliquot diluted 20-fold with 1´ Assay Buffer (50 ml in 950 ml of 1X Assay Buffer). Use the 1X Assay Buffer to zero the spectrophotometer. The A550/A565 ratio should be between 10 and 20.

Note: If the A550/A565 ratio remains less than 10, the substrate has not been sufficiently reduced and the

enzyme activity will not be valid. In this case refer to the Troubleshooting Guide (see Appendix).

Cytochrome c Oxidase Positive Control: Dissolve the vial in the volume of water specified in the instructions on the label/CofA. For the enzyme assay, further dilute the sample 10-fold with 1´ Enzyme Dilution Buffer and use 20–40 ml for each control reaction mixture. The sample may be stored at 2–8 °C for at least 3 weeks or frozen in aliquots at –20 °C.

Enzyme Sample: The best results are achieved when the enzyme activity is between 0.4–4.0 milliunits of

cytochrome c oxidase per reaction. For unknown samples, it is suggested to test several sample dilutions

to ensure the readings are within the linear range of the assay.

Reagents and Equipment Required but Not Provided.

  • Spectrophotometer
  • 1 ml Cuvettes
  • Analytical balance
  • Ultrapure water (³18 MW´cm resistivity at 25 °C)
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