The reaction mixture includes,
The extracted enzyme (250 microliter),
Tris-HCl (100 mM)
Betamercaptoethanol (1 mM)
L-Phenylalanine (50mM)
The total volume must be 1.5ml
The final concentrations of above mentioned reagents cannot be found by myself. Please help me to fix this issue, I really appreciate it.
Thank you
The solution can be prepared by mixing concentrated stock solutions of each reagent. The main limitation is the solubility of phenylalanine in water. You should be able to make a stock solution of at least 0.1 M.
1.5 ml reaction mixture
- 150 µl of 1 M Tris-HCl (pH chosen so that the final pH of the reaction mixture is obtained)
- 10.49 µl of a 1:100 dilution in water of beta-mercaptoethanol (143 mM)
- 750 µl of 0.1 M phenylalanine in water
- 340 µl of water
- 250 µl of extracted enzyme
My question: what is the initial concentration of the substrate L-phenylalaline and that of mercaptoethanol?
Joseph Kreit, Really sorry for not mentioning that in the question. I am going to use the concentrations follow mentioned
L-phenylalanine 50 mM
Mercaptoethanol 1mM
Dear Thathsarani
From your answer, I understood that the concentration of L-phenylalanine in your stock solution is 50 mM and that of mercaptoethanol is 1mM. It is right?
My question: what is the final volume of your assay? And what are the concentrations of these substances in the assay?
Dear Mr.Kreit,
These are the final concentration and volumes of the reaction mixture.
100mM Tris-HCl buffer
1 mM betamercaptoethanol
50mM phenylalanine
450 ul of the enzyme
I got these values directly from a paper.
Thank you
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating