Objectives: a) to quantify proteins in an unknown concentration of egg albumin (egg white) solution, and (b) to separate casein protein from milk    

Background: In the first part of this lab, the Biuret method will be used to identify and quantify proteins in an unknown concentration of egg albumin solution. Proteins are identified in this method due to the binding of a copper complex to peptide bonds in the protein chain giving a purple color. In order to quantify the concentration of the protein, you will prepare a standard curve using bovine serum albumin (BSA); a protein derived from cows.

In part 2, casein will be separated from milk by adjusting the pH of the milk to its isoelectric point and then centrifuging the precipitate. Proteins are least soluble at their isoelectric point since, at this point, their charges are neutral.

Materials:

  • Cow’s milk
  • Egg albumin solution
  • Bovine albumin serum (BSA)
  • Biuret reagent
  • 0.5 M HCL
  • 0.5 M NaOH
  • Distilled water
  • Test tubes
  • Test tube racks
  • Scale
  • Centrifuge
  • Stir plate
  • pH mater
  • Spectrophotometer
  • Weigh boat
  • Centrifuge tubes

Procedure:

Part 1. Protein quantification

  1. Label a set of test tube with numbers 1 through 6
  2. Pipet the amount of water indicated in the chart below into each tube
  3. Pipet the amount of BSA stock solution (10 mg/ml) indicated in the chart
  4. Pipet 1 ml of the egg albumin sample provided in two test tubes (to replicate reading)  
  5. Add 2 ml biuret reagent to all the test tubes and allow to sit for 15 minutes
  6. Use a spectrophotometer to measure the absorbance of each tube at 550 nm
  7. Using excel, graph the standard curve using the BSA concentration (x-axis) versus absorbance (y-axis). Show the equation of the standard curve and the R2 value
  8. Use the equation of the curve to calculate the protein concentrations of the egg solution (mg/ml) based on its mean (average) absorbance
Tube No.BSA Conc. (mg/ml)H2O (ml)BSA stock (ml)Biuret reagent (ml)Absorbance
1*01.002 
210.90.12 
320.80.22 
430.70.32 
540.60.42 
650.50.52 

*Number 1 tube will serve as your blank. Use it to calibrate the spectrophotometer.

Part 2. Protein separation

  1. Pour 100 ml milk solution in a 150 ml beaker
  2. Measure the initial pH. Observe the appearance of the milk at this pH
  3. Mix continuously on a stir plate while adjusting the pH gradually to 4.6 using 0.5M HCL and 0.5M NaOH as needed. Observe the appearance of the milk at this pH
  4. Pour the milk sample in two labelled 45 ml centrifuge tube (use a marker). Make sure the final weight of the tubes is the same
  5. Place in centrifuge and centrifuge at 3000 rpm for 5 minutes
  6. Remove the tubes and pour off the supernatant (top liquid)
  7. Use a spatula to remove the protein (casein) and weigh. Report the wet weight of casein recovered in g/1ml

Lab questions to address

  1. Explain the principle of the biuret test i.e. how it works
  2. Explain the concept of isoelectric point and why proteins precipitate at their isoelectric point
  3. Give examples of how the principle of isoelectric point precipitation or coagulation of proteins is applied in industry

Courtney Simons
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Courtney Simons is a food science professor. He holds a BS degree in food science and a Ph.D. in cereal science from North Dakota State University.
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