I want to prepare a standard solution of tyrosine for measuring protease activity and do a calibration curve but I don't know which is the best concentration to prepare and which step should I follow?
Hi there,
Tyrosine is one of the trickiest aminoacid as its limit of solubility in water is quite low, around 2,5mM. So your stock solution if prepared in aqueous solution as to be less than that. Now you have also to consider the range of concentration of tyrosine in the assay to make a stock which will be convenient from a pipeting point of view. For instance if possible think of a stock solution being 10x or 100x concentrated.
I want to do a standard curve with 50, 100, 150, 200 and 250 ug/ml and I don't know how much standard concentration and volume I have to prepare ?
2.5mM corresponds to 0.453g/L or 0.453mg/mL or 453µg/mL. So the highest concentration of your calibration is within the limit of solubility. If I were you I would prepare a 250µg/mL stock solution and work out dilution to generate the smaller concentrations. Knowing what volume you need for eacg calibration point you should be able to calculate the minimal volume you need of the stock solution...
You can easily prepare a 1mM L- Tyrosine standard by weighing up an appropriate amount and placing In the appropriate size volumetric flask, then bring to 50% if required volume with purified water. Then mix solution to dissolve in an 80 degrees C water bath. Once the tyrosines is dissolved you will bring to final volume with purified water. As an alternative Tyrosine can be dissolved in 10 mM HCl as it will just require mixing for a short time without heating. Either method used the Tyrosine stock standard solution can be stored for over several weeks refrigerated.
Whenever you prepare standard solutions, think about adsorbed water. The more hygroscopic your chemical, the bigger will be its effect. At least, store in a desiccator over silica gel, CaCl2 or similar.
You may also want to check the actual concentration of your chemical by some other means, like, for Tyr, UV-absorbance or nitrogen content.
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