SPECTROPHOTOMETRIC DETERMINATION OF THE CONCENTRATION OF A PROTEIN Purpose: The purpose of this lab is to determine the concentration of a protein using spectrophotometry. Discussion: Once a protein has been isolated through chromatographic techniques, it is useful to know how much of the protein was yielded from the cell. One technique to determine protein concentration includes the use of spectrophotometry. 1. How does spectrophotometry work? 2. How does Beer’s Law apply to spectrophotometry? 3. How does the Biuret reagent work, and how will it be used? 4. What is egg albumin protein? Objectives: In this lab, you will… 1. determine the concentration of egg albumin protein using spectrophotometric methods. Materials: Equipment: Chemicals: 5 mL Pipet Biuret reagent Pipet bulb 0.5000 M KCl 7 25 mL volumetric flasks 2.5 mg/mL albumin solution Spectrophotometer w/ cuvette adapter Unknown albumin solution 7 cuvettes Distilled water Laptop/LoggerPro Safety: Handle the chemicals with care, and always wear safety goggles. Procedure: 1. Clean and dry seven cuvettes and their caps. 2. Turn on the spectrophotometer. 3. Using Data Table 1, prepare the solutions in 25 mL volumetric flasks using the volumes listed for the standard 2.5 mg/mL albumin solution, Biuret reagent, and 0.5 M KCl solution. a. For solution 7, use 20.00 mL of unknown albumin solution and 5.00 mL of Biuret reagent. b. It is suggested that the standard albumin solution and Biuret reagent be pipetted with a 5 mL pipet. The solution can then be diluted up to the 25 mL mark with the 0.5 M KCl solution. Procedure (cont.): 4. Gently invert the volumetric flasks to mix the solutions. Transfer the solutions to cuvettes, but don’t fill above 0.5 cm of the top of the cuvette. Put the caps on the cuvettes. 5. On the spectrophotometer, pull the filter out to the orange filter. Adjust the wavelength to 540 nm. 6. Switch it transmittance mode. Insert cuvette 1 and adjust the transmittance to 100%. This will blank the spectrophotometer. Switch it to absorbance mode. 7. Run each cuvette (2-7) through the spectrophotometer. Record absorbances in the data table. Cleanup: Pour solutions down the drain with plenty of running water. Clean, dry and return all equipment used to its proper location. Data: Data Table 1: Contents of each cuvette to create standard solutions, their concentrations, and their absorbances at 540 nm to determine the concentration of an unknown albumin protein solution. Cuvette Volume of Volume of Volume of Concentration of Absorbance Number Standard Albumin Biuret 0.5 M KCl Albumin @ 540 nm (mL) (mL) (mL) (mg/mL) 1 0.00 5.00 20.0 2 4.00 5.00 16.0 3 8.00 5.00 12.0 4 12.00 5.00 8.0 5 16.00 5.00 4.0 6 20.00 5.00 0.0 7 X 5.00 X Analysis/Calculations: 1. Calculate the concentration of the albumin in each cuvette. 2. Construct a standard curve using the concentration of albumin (mg/mL) and the absorbance. 3. Use the equation for the standard curve and the unknown albumin absorbance to determine the concentration of the unknown albumin solution. Error: 1. Ask your instructor for the true value of the unknown albumin solution. 2. Calculate absolute error and percent error.