Determination of absorbance values of solutions by spectrophotometer

Summary

absorbance

Principle

In protein purification experiments, the activity of b-glucuronidase (GUS) will be measured by using p-nitrophenyl b-D-glucuronide as the substrate, and the p-nitrophenol released from the reaction of adding enzymes shows yellow color under alkaline condition, and the absorbance at 415 nm can be measured, and the amount of the reactants produced can be calculated by using the Beers Law. The amount of reactants produced can be calculated using the Beers Law. In this experiment, the A415 of p-nitrophenol was measured using a known concentration of p-nitrophenol to determine the extinction coefficient of p-nitrophenol under the conditions of GUS activity measurement.

Appliance

Content determination

Operation method

Determination of absorbance values of solutions by spectrophotometer

Materials and Instruments

【Materials】p-Nitrophenol10×GUS assay buffer (0.5 M Na phosphate, pH 7.0; 1% Triton X-100; 0.1 M β-mercaptoethanol) GUS terminator (2.5 M 2-amino 2-methyl propanediol, dissolved in water, pH 11) GUS termination solution (2.5 M 2-amino 2-methyl propanediol, soluble in water, pH 11)

Move

1) Prepare 5 mM p-nitrophenol stock solution: Weigh _____ mg of p-nitrophenol in a 400 mL beaker, add 25 mL of 10×GUS assay buffer, add water to about 200 mL, stir to dissolve, add water to 250 mL and mix well. Please prepare three portions.2) Preparation of 50 μM p-nitrophenol solution: Dilute the stock solution with 1×GUS assay buffer.(3) Take 1 mL of 50 μM p-nitrophenol solution in a test tube, add 0.4 mL of 2.5 M 2-amino2-methyl propanediol solution, mix well and then measure A415. Take another 1 mL of 1×GUS assay buffer, add 0.4 mL of 2.5 M 2-amino 2-methyl propanediol solution, mix well. A415 was also measured by adding 0.4 mL of 2.5 M 2-amino 2-methyl propanediol solution to 1 mL of 1×GUS assaybuffer, mixing well, and using it as a blank.4) Determine the extinction coefficient of p-nitrophenol under these conditions by Beer's Law.

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