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.
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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:
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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 |
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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
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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