I'd like to know if anyone can provide me with a protocol for traditional catalase enzymatic assay in a 96 well microplate, for cell line and/or C.elegans spectrophotometric measurements.
Thanks!
Hi Santiago, I have this one from an ELISA kit, maybe it will help you.
You can use the classical Aebi (1984) method.
Here in the lab, we usually use the following final concentrations:
- 50 mM phosphate buffer containing 0.5 mM EDTA (pH 7.0–7.2)
- 10 mM hydrogen peroxide
Then, read the microtiter plate at 240 nm for 40 s at 8 s intervals in the kinetics mode.
Two important issues here: (i) not all plates are transparent at 240 nm, be sure to use UV-transparent plate for readings; (ii) correct the pathlength depending on the final volume in each well, since you will be using the molar extinction coefficient of hydrogen peroxide = 0.04 mM-1 cm-1 (note the cm-1 referring to the pathlength).
Article [13] Catalase in vitro
Thank you all for your quick answers. Aebi's classical method seems to be the simplest and more economic assay, but I'll keep reading your comments or suggestions!
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