Can anyone suggest a good protocol for catalase assay for root tissue homogenate. I homogenised the root with phosphate buffer and took the supernatant, to it added hydrogen peroxide phosphate buffer, this was taken as experiment, and hydrogen peroxide free phosphate buffer with plant extract was taken as control.Some one please tell me what should i do next, to find out the catalase activity i.e about spectral analysis and so on...
The below protocol have been used for both leaf and root catalase activity estimation:
The samples were ground at 4 °C in a porcelain mortar with homogenization buffer containing 100 mM K2HPO4, pH 7.0, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 0.3% ethanol. The homogenization was carried out using 5 mL of buffer per g fresh sample weight. The homogenate was centrifuged at 3300× g for 5 min at 4 °C. The resulting supernatant was subsequently used for both CAT activity and protein concentration estimation. Catalase was assayed by its depletion of H2O2.
The standard CAT assay consists of mixing (in a 30 °C water bath) 0.85 mL of sample (proper dilution of the homogenate supernatant), 0.05 mL of the homogenization buffer, and 0.1 mL of 0.09 M H2O2 stock solution (made fresh in homogenization buffer). The linear absorbance decrease during the assay mixture was measured at 240 nm in a spectrophotometer. The absorbance decrease rate of consumed H2O2 was converted to CAT units (U) from a pure CAT standard curve.
Catalase was assayed by its depletion of H2O2
Please Check the below mentioned paper:
Aebi, H. Catalase in Vitro. Methods Enzymol. 1984, 105, 121
–126.
- Badges
- Science topic