EXERCISE 8A Name Isolating purifying and characterizing proteins Day One How can lactalbumin be separated from the other molecules in milk Objectives After completing this exercise you should be able to Explain why scientists are interested in purifying and characterizing the proteins and other biomolecules found in living organisms Describe how size exclusion chromatography separates a mixture of biomolecules Describe and use the following techniques to separate lactalbumin from the other biomolecules in nonfat milk differential solubility centrifugation ultrafiltration and size exclusion chromatography Describe and use optical absorption at 280 nm A280 to estimate the protein concentration in an elution profile from a chromatography column Prelab Before you come to lab read this entire exercise You must also answer all questions and complete all assignments on the first 8 pages of this exercise Your instructor will give you directions on when and where to turn in your work Cells are composed of thousands of biomolecules acting and interacting in many complex ways Most of these biomolecules are present in extremely small amounts Therefore in order to study the structure and function of cellular biomolecules molecular biologists must overcome two obvious problems 1 The molecules must be separated or isolated from all of the other biomolecules present in the cell In other words a single type of molecule must somehow be plucked out of the rich cellular soup made of thousands of different kinds of molecules 2 Enough molecules of each type must be isolated for study A few dozen molecules or even a few hundred molecules are not enough to work with A scientist needs enough of the substance to be able to run a variety of biochemical tests in order to determine the structure and function of the biomolecule Often the molecules that a biologist wants to isolate and study are proteins 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 four lab periods you will study several important techniques that molecular biologists use to isolate specific proteins from a complex mixture of biomolecules You will use these techniques to separate a protein called lactalbumin from the other biomolecules found in nonfat milk At several points during the procedure you will save samples of the purification fractions These fractions will be analyzed in order to determine the total protein concentration of each fraction In addition each fraction will be analyzed using SDSPAGE electrophoresis in order to determine the number of proteins present in each fraction and their molecular weights Finally you will identify the various proteins present in each purification fraction based on their molecular weights Exercise 8A Austin Community College BIO 1406 Laboratory Manual 12th Ed 2006 8A 1 Schedule for Exercise 8 Part A Carry out several purification steps in order to separate lactalbumin from the other biomolecules present in nonfat milk Part B Determine the total protein concentration of each purification fraction Part C Calculate how much of each purification fraction should be loaded onto your SDS PAGE gel and then load run and stain your gel Part D Analyze your SDS PAGE gel in order to determine which proteins are present in each purification fraction Also evaluate your success in isolating a sample of pure lactalbumin Main steps involved in purifying lactalbumin 1 Set aside a sample of nonfat milk to assay later 2 Precipitate the casein milk proteins 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 3 Set aside a sample of the pellet of precipitated proteins to assay later 4 Remove any remaining precipitated proteins from the supernatant whey using ultrafiltration 5 Set aside a sample of the whey to assay later 6 Separate lactalbumin from the other proteins that remain in the whey using size exclusion chromatography 7 Set aside the five chromatography fractions most likely to contain lactalbumin to assay later 8 Prepare a standard curve for the Bradford assay using solutions of known protein concentration 9 Use the Bradford assay and your standard curve to determine the protein concentrations of the 8 milk fractions that you set aside on Day1 10 Calculate the amount of each milk fraction that should be loaded into your SDS PAGE gel so that each lane contains an appropriate amount of protein 11 Load run and stain SDS PAGE gels 12 Analyze your gel to determine the number of different proteins that are present in each milk fraction and the molecular weights of these proteins 13 Use your gel analysis to evaluate how effectively you isolated and purified lactalbumin from the other components of milk Exercise 8A Austin Community College BIO 1406 Laboratory Manual 12th Ed 2006 8A 2 Which molecules are found in milk 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 and their molecular weights are listed in the following table Milk Protein Molecular Weight daltons lactalbumin 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 The 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 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 lactalbumin which is secreted into the milk 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 and the immunoglobulins are antibodies that help protect the newborn from