I am trying to do some stress related assays on plants and have limited amount of tissue. When searching literature for catalase assay, I found protocols where tissues are used for doing catalase assay. Can anyone suggest me a protocol where total protein can be used instead of tissue
I think that you can use a standard protocol although you use total protein (an extract, I suppose...). You only need not to degrade the protein.
You can precipitate total proteins from plant extract by cold (at -20 oC during night) acetone (one half of extract) and after cenntrifugation you can obtain protein pellet and then you can dissolve the pellet in a suitable buffer. You can use it as enzyme source.
The CAT reaction solution (3 mL) contained 50mM phosphate buffer (pH 7), 5.9 mM H2O2 and 0.1 mL enzyme extract. The reaction was initiated by adding the enzyme extract. Changes in absorbance of the reaction solution at 240 nm were read every 20S. One unit of CAT activity was defined as "An absorbance change of 0.01 units per min".
The activity of each enzyme was expressed on protein basis. Protein concentration of the crude extract was measured by the method of Bradford (1976).
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