Someone please help me in plotting standard curve for Tyrosine. I made different concentration of tyrosine including 0.05-0.3 mg/ml by dissolving in HCl. Then using 200 microlitre of different concentration of tyrosine, I added 1 ml of 0.5% casein. (Later on, I also used 1% casein) then incubated at 37 degrees for 30 min and then stop the reaction by adding 200 microlitre of 40% TCA. After 10 min, I centrifuged it at 9000 rpm for 2 min and took OD reading at 280 nm. But unfortunately, I am unable to plot standard graph. Where am I making the mistake. Please guide me.
Thank you
What is the rationale for this method? You have a solution of tyrosine. You could just measure its absorbance.
Instead you add a protein (casein), which also contains aromatic amino acids including tyrosine. The HCl will probably cause some hydrolysis of the casein, releasing some of these amino acids. Then you precipitate the casein with TCA. The casein + TCA step seems unnecessary and problematic. Additionally, some of the free tyrosine will probably get caught up in the large casein pellet, causing an underestimation of the added free tyrosine.
The aim of the standard curve is to estimate the amount of liberated tyrosine. You do not have to treat tyrosine in the same way as you do to estimate the casein hydrolysis, you will get a lot of Impurities in your solution. all you have to do to measure the absorbance of the standard solution of tyrosine with various concentrations by direct reading at 280 nm, or by lowry method between 550-750 nm according to the expected concentrations.
Although the original producer of casein digestion of (Kunitz, 1947) implicate treatment of tyrosine with the same protocol that casein has treated.
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