I would like to make catalase activity standard curve, but i still fail.
I would like to know protocol of making catalase activity standard curve.
Do you mean oxygen production rate as function of [H2O2], pH, temperature etc.? Many years back as undergrad student I did that with spinach leaf extract (20 g leafs ground with sand diluted to a final volume of 100 ml with distilled water and filtered through cheese cloth).
3 ml of this extract (or of further diluted extracts to measure the effect of enzyme concentration) are added to 20 ml 0.2 M phosphate buffer (pH 7.2 if you are not measuring pH dependence) from a funnel with ground glass valve into a 3-neck bottle thermostated (to 25 °C if you are not measuring the effect of temperature). The second neck is connected to a pneumatic bath where the released oxygen is captured in a graduated test tube. The third neck of the reaction vessel is used for pressure release when you add the reagents. The contend of the vessel is magnetically stirred.
Reaction is started after 5 min temperature equilibration with 10 ml H2O2 (3% if you are not measuring the effect of substrate concentration). Timer is started when the first bubble enters the test tube, time is 1 min unless you measure the effect of time. Under the standard conditions mentioned, you should get about 10-15 ml O2 per min, adjust the amount of leaf extract if necessary.
See links for measuring the activity of Catalase.
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0ahUKEwiDp93A2p_QAhXC7oMKHZ59C38QFggbMAA&url=https%3A%2F%2Fwww.researchgate.net%2Ffile.PostFileLoader.html%3Fid%3D5660d7be6225ff8ffe8b4567%26assetKey%3DAS%253A302727475728384%25401449187262027&usg=AFQjCNEc892_sS_NBBZF2ZFnp8U0_mnBJg
http://www.biologyjunction.com/enzyme_catalysis.htm
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