I've read several works that assess catalase assay from samples by spectrophotometry based on Aebi (1984). Based on this, some works employ a microplate for this determination, and this is what I want to do with eukaryotic cells extract/C. elegans extract (i.e.: Article Development of a 96-well based assay for kinetic determinati...
,Article Rapid Kinetic Microassay for Catalase Activity
)I have a couple of questions regarding this:
- Is it necessary to include a purified catalase concentration curve in this determination or this is only applied when validating a techinique? This method has been validated as I showed above.
- Can anyone provide to me a detailed protocol of this so I can reproduce it at my lab?
- How can I perform the measurements at optimal temperature if my spectrophotometer does not have a thermal regulation?
Looking forward for your answers!
Here are a few RG discussions that may be helpful:
https://www.researchgate.net/post/A_protocol_for_catalase_and_superoxide_dismutase_assay2
https://www.researchgate.net/post/In_the_catalase_assay_why_use_the_bradford_regent_for_get_a_absorbance_Why_use_only_the_bradford_regent_for_idetify_the_catalase_enzyme
Hi,
1. No, it is not necessary to have include a purified catalase concentration curve. You can use the molar extinction coefficient of H2O2 to actually calculate how many moles of H2O2 are consumed per unit of time and, thus, express catalase units.
2. I do not have a detailed protocol, but a important issue when using microplates is the pathlength. In cuvette, the pathlength is defined by the dimension of the cuvette. In microplates, however, the pathlength varies and depends of the volume in which well. Since most of molar extinction coefficients available in the literature are expressed as per cm-1 (the "standard" cuvette width is 1 cm), you must correct for it.
3. Aebi 1984 recommends to make short readings (~30 seconds). Thus, if your microplate reader does not control temperature, you can place all reagents in a water bath at the desired temperature and quickly dispense them in the plates.
Hello
Some of necessary recommendations have made above.
I used Goth 1991 in microplate to assay plasma CAT activity in fish:
A simple method for determination of serum catalase activity and revision of reference range. Clinica chimica acta 196 (2-3), 143-151, 1991
But you need validate it using catalase standard.
You should note that CAT assay is time-dependent and you must take into acount the pippeting tome and lags of reading multiple wells by the apparatus
Thanks a lot for your detailed suggestions. 10 mM hydrogen peroxide, Aebi's reagents, times and wavelenght and Schellhorn's microplate adaptated protocol would be my choice for cell culture evaluation of catalase activity
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