What are the main steps involved in isolating ?-lactalbumin from milk?

BIOL 1406

PreLab 8a.1

What are the main steps involved in isolating α-lactalbumin from milk?

Cells are composed of thousands of different biomolecules. Most of these biomolecules are present in extremely small amounts. Therefore, in order to study the structure and function of a specific biomolecule, molecular biologists must overcome two obvious problems:

1) The biomolecule of interest must be separated, or isolated, from all of the other biomolecules present in the cell.

2) A large enough sample must be isolated so that scientists can run the various tests needed to determine the structure and function of the biomolecule.

Often, the biomolecule that a biologist wants to isolate and study is a protein. This is not surprising because proteins are the most abundant and versatile organic molecules found in cells. Cells are able to make tens of thousands of different proteins, each with its own unique structure and function.

During the next 4 lab exercises, you will attempt to isolate the protein α-lactalbumin from milk. Milk is a complex mixture of biomolecules secreted by the mammary cells of mammals. Like cells, milk is mostly water. It also contains various inorganic ions as well as many organic molecules including lipids, carbohydrates, and dozens of different proteins. Major proteins found in milk, along with their molecular weights, are listed in the following table:

Major Milk Proteins	Molecular Weight (daltons)
α-lactalbumin β-lactoglobulin various caseins blood serum albumin lactoferrin various immunoglobulins	14,437 18,000 ~19,000-30,000 68,000 87,000 ~160,000-1,000,000

Ribbon diagram of alpha-lactalbumin

Caseins are found in large protein complexes called micelles, which contain many phosphate groups and also bind calcium ions. These protein complexes make up about 80-90% of the total protein found in milk and supply the newborn with calcium, phosphorus, and the amino acids needed for protein synthesis.

Blood serum albumin is a protein that leaks into the milk from the bloodstream, where it helps maintain the proper osmolarity of body fluids. Lactoferrin has antibacterial properties, while immunoglobulins are antibodies that help protect the newborn from disease. The function of β-lactoglobulin is not known.

Another nutrient found in large quantities in milk is lactose, or milk sugar, which provides readily available energy to the newborn. Lactose is made when a glucose molecule and a galactose molecule are joined by the action of the lactose synthase enzyme complex. This complex is made up of the enzyme galactosyltransferase, which remains in the mammary cells, and α-lactalbumin, which is secreted into the milk.

Although α-lactalbumin makes up only about 2-5% of total milk proteins, your goal in this lab activity is to separate it from all of the other molecules found in milk.

Schedule for Exercise 8
Part A:	Carry out several purification steps in order to separate α-lactalbumin from the other biomolecules present in nonfat milk. Save samples at various stages of the purification process for analysis later.
Part B:	Determine the total protein concentration of each sample saved during the purification process.
Part C:	Calculate how much of each purification sample should be loaded onto your SDS-PAGE electrophoresis gel; and then load, run, and stain your gel.
Part D:	Analyze your SDS-PAGE electrophoresis gel in order to determine which proteins are present in each purification sample. Also, evaluate your success in isolating a sample of pure α-lactalbumin from nonfat milk.

Main steps involved in purifying α-lactalbumin

Set aside a sample of nonfat milk to assay later
Precipitate the caseins using heat and low pH. The other milk proteins should remain in solution. Centrifuge the heat and acid treated milk to separate the precipitated casein proteins from the soluble proteins.
Set aside a sample of the pellet to assay later.
Remove any remaining precipitated proteins from the supernatant (whey) using ultrafiltration.
Set aside a sample of the whey to assay later.
Separate the proteins that remain in the whey using size exclusion chromatography.
Set aside the five chromatography fractions most likely to contain the highest concentrations of α-lactalbumin to assay later.
Prepare a standard curve for the Bradford assay using solutions of known protein concentration.
Use the Bradford assay and your standard curve to determine the protein concentrations of the 8 milk fractions that you set aside on Day 1.
Calculate the amount of each milk fraction that should be loaded into your SDS-PAGE electrophoresis gel so that each lane contains an appropriate amount of protein.
Use SDS-PAGE electrophoresis to determine the number of different proteins that are present in each milk fraction and the molecular weights of these proteins.
Use your gel analysis to evaluate how effectively you isolated and purified α-lactalbumin from the other components of milk.

Your Turn
What is the most abundant molecule found in milk?	Check your answer.
What is the most abundant group of proteins found in milk?	Check your answer.
Which protein will you attempt to isolate from milk?	Check your answer.
How will you remove the caseins from your original milk sample?	Check your answer.
After removing the caseins from milk to form whey, how will you separate the various proteins that are left in the whey?	Check your answer.
How will you determine the protein concentration of the milk samples saved during the purification process?	Check your answer.
How will you determine the number and size of the proteins present in each milk sample that was saved during the purification process?	Check your answer.

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