No Whey! Milk Protein Content Doesn't Change...Or Does It?

Abstract

Many people you know probably have an opinion about the kind of milk they like to drink—some like it thin and refreshing, others like it thick and rich. Milk can be bought with different fat concentrations, but other than that, it's all the same. Or is it? This science fair project raises a few interesting questions about the other contents in milk. Do all milk products have the same protein concentrations? Do cows produce different types of milk during different stages of lactation? There's only one whey to find out—give this science fair project a try!

Objective

To determine total protein concentration, casein concentration, and whey protein concentration of cow milk samples retrieved from cows during different stages of lactation.

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Last edit date: 2020-01-12

Introduction

Milk—not only does it taste good with fresh cookies or poured over a bowl of cereal, but it is also critical for the healthy development of all young mammals, including humans. To feed their young, all female mammals produce milk after birth. This period of milk production is called lactation. There are four stages of lactation. Colostrum is secreted for the first few days, and then transitional milk until the end of the second week. During full lactation, mature milk is made; and then around the end of lactation, during the fourth stage, involutional milk is made. Are there differences in the milk produced between these stages of lactation?

First, consider that there are two main types of proteins in milk: caseins and whey. Caseins have specific amino acid compositions that make them important for the growth and development of the nursing young. All other proteins in milk are known as whey proteins. The two most abundant whey proteins in milk are beta-lactoglobulin and alpha-lactalbumin. Other whey proteins include: immunoglobulins, serum albumin, and various enzymes, hormones, growth factors, nutrient transporters, and disease-resistance factors.

In this science fair project, you'll determine whether the different stages of lactation affect the protein composition of cow's milk. Does the total protein level change? How about the quantity of casein versus whey protein? If you don't have access to a dairy from which to obtain fresh milk samples, you can try one of the variations, at the end of the Experimental Procedure, about protein concentrations in different types of milk. Regardless of which question you choose to examine, you'll use the Bradford Assay to quantitatively determine the protein concentrations. The Bradford Assay is based on the binding specificity of the dye Coomassie Brilliant Blue-G250 for proteins, but not for other molecules. Coomassie maximally absorbs light at the wavelength of 470 nanometers (nm). However, this changes to 595 nm when the dye is bound to a protein. The Bradford Assay takes advantage of this by mixing protein samples with Coomassie and then measuring the amount of absorption, using an instrument called a spectrophotometer, at 595 nm. The greater the amount of absorption at 595 nm, the larger the concentration of protein in the sample. Figure 1, below, shows the electromagnetic spectrum and where Coomassie bound to protein absorbs light (at 595 nm) compared to where Coomasie alone absorbs light (at 470 nm).

To convert the amount of absorption by a sample containing an unknown concentration of protein into an exact protein concentration, first a set of solutions with known protein concentrations are measured. The absorption of the known samples are graphed with the protein concentration on the x-axis and the absorption on the y-axis to form a standard curve. The standard curve can then be used to determine the quantity of protein in an unknown sample by either reading the graph to estimate the protein concentration at a given absorption, or by finding the equation that describes the standard curve and using that equation to solve for the protein concentration, x, of an unknown sample that has a measured absorbance of y.

To answer the questions in this science fair project, you'll measure the total protein concentration in each milk sample, using the Bradford Assay. Then you'll separate the milk samples into their casein- and whey-component parts and measure the concentration of each type of protein. Will any of the concentrations change? Break out the milk, the spectrophotometer, and your lab gear to find out the answer. And when the hard work is over, treat yourself to a nice, tall, cold glass of milk, caseins and whey included!

Terms and Concepts

• Lactation
• Colostrum
• Involution
• Casein
• Whey
• Bradford Assay (also called Bradford Method or Bradford Test)
• Coomassie Brilliant Blue-G250
• Spectrophotometer
• Standard curve

Questions

• What are the molecules in milk?
• What are the differences between the four stages of lactation?
• How does the Bradford Assay work?
• What are the limitations of the Bradford Assay for estimating protein?
• How does a spectrophotometer work?
• How can a standard curve be used to make estimations?

Bibliography

• Brown, K. (2013, October 2). Bradford Test for Protein. Retrieved October 28, 2013, from https://www.tracy.k12.ca.us/sites/emckay/Shared%20Documents/IB%20Bio%20I/Labs/Bradford%20Test%20for%20Proteins%20LAB20132014.pdf
• Hurley, W.L. (n.d.). Milk Composition: Proteins. Retrieved January 12, 2009, from the University of Illinois' website: http://classes.ansci.uiuc.edu/ansc438/Milkcompsynth/milkcomp_protein.html
• Prentice, A. (December 1996). Constituents of Human Milk. Retrieved January 17, 2009 from the United Nations University website: http://www.unu.edu/unupress/food/8F174e/8F174E04.htm
• Thermo Fisher Scientific Inc. (2007.) How To Use a Protein Assay Standard Curve. Retrieved July 1, 2009, from http://www.thermo.com/eThermo/CMA/PDFs/Articles/articlesFile_7176.pdf

Materials and Equipment

Note: This science fair project requires access to laboratory equipment and supplies, including a spectrophotometer.

These items can be purchased from Carolina Biological Supply Company, a Science Buddies Approved Supplier:

• Cuvettes (50). Note: You should confirm that these cuvettes will work with the specific spectrophotometer that you will be using. Also, make sure the spectrophotometer does not already have cuvettes that you can use.
• Microcentrifuge tubes, 1.5-mL size
• Transfer pipettes, capable of dispensing in 1-mL quantities, up to 5 mL
• 5 mL test tubes (12)
• 0.1 M hydrochloric acid. Note: If you are ordering this chemical through Carolina Biological Supply Company, the chemical must be ordered by a teacher and shipped to a school or business address, so plan accordingly.
• pH meter

You will also need to gather these items:

• Cow milk samples from a local dairy
  • 5-mL samples (3 for each of the four stages of lactation, for a total of 12 samples)
• Refrigerator
• Spectrophotometer
• Distilled water (1 gal)
• Microcentrifuge
• Micropipettes
• Micropipette tips
• Bradford Assay reagents. Consult your specific lab's protocol for specific reagents needed. At minimum, the following items will be required:
  • Bovine Serum Albumin (BSA) for preparing a stock solution and further dilutions to create a protein concentration standard curve
  • PBS, or some other buffer, for preparing samples
  • Coomassie, or some other specifically formulated Bradford dye reagent
• Lab notebook
• Graph paper

Experimental Procedure

Important Notes Before You Begin: The details of this experiment will depend on the type of spectrophotometer and other equipment available to you. For this reason, the procedure below only outlines the general steps necessary to carry out this science fair project. You should either be familiar with all the procedures below from prior laboratory or classroom experience, or have a mentor (such as your science teacher) who can help guide you. The references listed in the Bibliography and our guide to Chemistry Lab Techniques can also be consulted if you have questions about a procedure.

Collecting Milk Samples

1. Work with a local dairy to obtain your milk samples. If you are gathering the samples yourself, follow the directions of the dairy workers to ensure the health and safety of the cows.
   1. You should have three separate samples from each of the four lactation stages, for a total of 12 samples.
2. Label and store all milk samples in a refrigerator until you are ready to begin your experiment.

General Notes About Preparing and Measuring Samples for the Bradford Assay

Note: Volumes and exact reagents will depend on the spectrophotometer, size of the cuvettes, and Bradford Assay kit you chose to use. Consult your specific lab's protocols and the Bradford Assay kit for step-by-step instructions.

1. Treat all samples in the same way, including:
   1. Diluting all samples in the same buffer; PBS is a good choice.
   2. Using the same type of cuvettes with the same final volume for all samples.
   3. Adding the same quantity of Coomassie for all samples.
2. Make sure you know how to use the spectrophotometer before you get your samples ready.
   1. Consult the spectrophotometer manual if you have any questions, or find a knowledgeable mentor.
   2. Always use a blank (a sample containing only buffer and no protein) to zero the spectrophotometer before taking absorption readings
   3. Make sure the spectrophotometer is set to excite at a wavelength of 595 nm.
3. Take absorption readings from your standard curve solutions and all your experimental samples during the same session. This ensures that there are no added variables introduced.
   1. If you need to make additional readings of experimental samples during another session, also make up and measure the absorption of a new set of standard curve solutions. Only use the standard curve created during the same session to estimate protein quantities in a given set of experimental samples.

Preparing the Standard Curve Samples

1. Using micropipettes to measure accurately, make a set of known protein concentration solutions for creating the standard curve.
   1. Consult your lab's protocol for a recommended range of concentrations for the solutions. Use no less than five concentrations to create your standard curve.
2. Solutions can be made using a stock solution of Bovine Serum Albumin (BSA).
   1. To calculate the amount of the stock solution needed to create each dilution, you'll first need to know:
      • What the concentration of the BSA stock solution is.
      • What final volumes you want for each of your dilutions (100 ul is sufficient for most spectrophotometer setups).
   2. Then use Equation 1, below, to solve for the volume of stock solution needed to create each dilution:

Equation 1:

$$V_1 = {\frac{C_2 V_2}{C_1} }$$

• V₁ is the volume of stock needed to create the dilution, in ul.
• C₂ is the desired final concentration of the dilution, in mg/uL.
• C₁ is the concentration of the stock solution, in ul.
• V₂ is the desired final volume of the dilution, in ul.

Preparing the Milk, Casein, and Whey Samples

1. Mix each milk sample by shaking it up and down. Reserve 1 mL of each milk sample to use to measure total milk protein concentration. The remaining 4 mL of each sample will be separated into casein and whey solutions to measure the concentration of each category of protein in the individual samples.
2. Pipet 4 mL of each milk sample into a culture tube. Make sure the tubes are accurately labeled so that you know which sample is which.
3. Lower the pH of each sample to approximately 4.6. This will allow the milk to be separated into its casein and whey protein components.
   1. Add 0.1 M hydrochloric acid slowly, drop by drop, to the first sample. Mix often and monitor the sample's pH, using a pH meter, until the sample reaches a pH close to 4.6.
      • Note: Make sure you know how to use the pH meter before doing this step. Remember to calibrate the probe prior to starting and to rinse off the probe with distilled water between samples.
   2. Repeat the process for the other 11 samples.
4. Once all milk samples are approximately at pH 4.6, transfer 1 mL of each sample into a labeled microcentrifuge tube.
5. Centrifuge all 12 sample tubes in a microcentrifuge at 14,000 revolutions per minute (rpm) for 5 minutes (min.).
6. After the centrifuging, the milk samples will be separated into three distinct layers, as seen in Figure 2, below. The cream is the top layer, the whey proteins are in the middle layer, and the caseins are in the bottom layer.

7. Separate the milk's components by carefully pipetting the caseins into one set of labeled microcentrifuge tubes, and the whey proteins into another set of labeled microcentrifuge tubes. Discard the milk's cream.
   1. Troubleshooting: If the caseins form a pellet and cannot easily be pipetted out, remove and save the whey first, then pipette away the cream. Gently wash the casein pellet with a little PBS before reconstituting the pellet in 20 ul of PBS.
8. At this point, you should have a total of 36 samples:
   1. 12 whole milk samples
   2. 12 whey samples
   3. 12 casein samples
9. Make 1/50 dilutions of each sample and prepare them for the Bradford Assay, according to the kit, or your local lab's protocol.
   1. Note: 1/50 dilutions are needed because the protein concentration in the samples are too high to be in the linear range of the assay.

Running the Samples and Analyzing the Results

1. Using the spectrophotometer, measure the absorbance of each sample. Each sample should be measured in a cuvette, as shown in Figure 3, below.
   1. Start by calibrating the spectrophotometer, using the blank.
   2. Measure all the standard curve dilutions and then move on to the 36 milk samples.
   3. Record the absorbance of each standard curve and experimental sample in your lab notebook.

2. Using a graphing program, like Microsoft® Excel®, create a standard curve by plotting the protein concentration and absorbance of each standard curve dilution on the x- and y-axis of a graph, respectively.
3. Use the graphing program to determine the equation that best fits the standard curve.
   1. Note: Consult the references in the Bibliography if you need further instructions on how to do this and subsequent standard curve steps.
4. For each experimental sample, plug the sample's absorption into the equation (as y) and solve for the protein concentration (x). Record each protein concentration in your lab notebook.
5. Find the average of similar samples. In the end, you should have 12 protein concentration averages. Three (whole milk, whey only, and casein only) for each of the four lactation stages.
6. Graph the averages. Do the total protein concentrations change across lactation stages? If so, which type(s) of proteins change quantity: the caseins, the whey proteins, or both?

If you like this project, you might enjoy exploring these related careers:

Variations

• Evaluate the protein concentrations from different percentages of store-bought milk and milk products, like cream and buttermilk.
• Compare the protein concentrations from milks from different species of mammals. Some grocery stores carry sheep or goat milk, in addition to cow milk.

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