Hi
I would like to know how to construct standard curve for azocasein assay? I know that tyrosine is usually used for the standard curve of protease assay. The tyrosine standard measured spectrophotometrically at 660 nm by adding with FC phenol reagent but in azocasein assay, enzyme activity is measured at 440 nm. So I suppose tyrosine cannot be used for the standard curve.
And another question is in azocasein assay, I notice that the last step involves centrifuging the enzyme assay mixture and and supernatnat is mixed with NaOH. In one journal, they reported they mixed with just water. Which one is right? What is the purpose of mixing with either water of NaOH?
Article Azocasein assay for alkaline protease in complex fermentation broth
Article Comparison of Methods for Assay of Fecal Proteolytic Activity
Check out this interesting article.
BioMed Research International
Volume 2016, Article ID 8409183, 6 pages
http://dx.doi.org/10.1155/2016/8409183
Hello
you can use tyrosine as standard in either of two methods ,both methods measure tyrosine produced upon protease action on casein where the first method measured a color of the product upon reaction with FC reagent. While the second one measure the product spectroscopy.
I think that it is better to use the buffer rather than water in regard to your second question since you will keep the pH of the solution ,since the change in pH can cause a shift in the wavelength of the absorbance .
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