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

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:

• 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.

Figure 2. The three layers of milk sample (cream, whey proteins, and casein) after milk's pH had been lowered to around 4.6, and then centrifuged at 14,000 rpm for 5 min.

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.

Figure 3. To use the spectrophotometer to measure you samples, you will be putting your samples in cuvettes, as shown here. (Photo courtesy of Bio-Rad Laboratories, Inc.)

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?