Isolation and Characterization of alpha-Lactalbumin

1. Isolation and Characterization of alpha-Lactalbumin Experiment#4 Week#1--Protein purification using Gel Filtration

2. Protein Separation/ Purification • Purpose: examine techniques in purifying -Lactalbumin, a constituent in milk • alpha-Lactalbumin: • MW= 15,500 Daltons • contains 129 amino acid residues • an essential component of the lactose synthase system

3. Purpose (continued) • Milk contains a variety of proteins….. -lactalbumin, -lactoglobulin, albumin, immunoglobulins, and caseins • Is -lactalbumin the predominant protein? • Protein will be assayed by • Isolating/purifying the whey with gel-filtration (week#1) • characterizing the protein using gel electrophoresis and UV spectrophotometric scans • estimating the protein concentration using the Bradford Reagent Spectrophotometric assay

4. Protein separation • Benefits: • provides the ability to identify cellular proteins • could be very valuable in cancer research • each type of cancer produces its own protein biomarkers • (current technology is not accurate and sensitive enough to detect most of these proteins)

5. Before proteins can be examined they must be removed from their source…. • Methods • Osmotic lysis • suspending cells in a high ionic strength solution causing the water inside the cell to diffuse through the membrane • gentle grinding in homogenizers • cells are forced against the glass walls under the pressure of the piston, releasing the cell components into the surrounding aqueous environment • ultrasonic wave cell disruption • the vibration from the ultrasonic waves disrupts the cell membrane, releasing the cell components • ultra-centrifugation • subjecting the cells to an intense centrifugal force causing sedimentation of cell organelles

6. Protein separation • Alpha-Lactalbumin from a sample of non-fat milk was removed in the following manner……. Nonfat Milk Centrifuge at 16,000 x g (step completed) Removes caseins and phosphoproteins Sediment (white) Discard Supernatant Change pH to 4.6 (using HCl) Heat to 40o C with stirring for 30min Centrifuge at 12700 RPM for 30 min Sephadex Chromatography Supernatant Clarify by filtering (whey) Sediment (white) Discard Purified alpha-Lactalbumin Characterization by electrophoresis, Bradford Assay, and UV analysis

7. Protein separation • After isolation, the protein must be kept stabilized. Control the following factors: • ionic strength • pH • temperature • presence of stabilizing ions (Na+, K+, Ca+2, Fe+2) • absence of deleterious ions (Ag+, Cu+2, Pb+2, Hg+2)

8. After isolation, protein is not in its purest form and must be further purified (many commercial proteins have a maximum purity of only 85%) • PROTEIN PURIFICATION TECHNIQUES • Ion-exchange Chromatography • ionic solutes display reversible electrostatic interactions with a charged stationary phase • Adsorption Chromatography • relies on specific interactions between the solute molecules and reversible binding sites on the surface of the stationary phase • Gel-Filtration • aka Size-Exclusion Chromatography or Column Chromatography • solutes passed through a column of porous stationary phase separating based on molecule size • Affinity Chromatography • separation based on biological interactions with ligands on stationary support---very specific • Under Development--Fast Protein Liquid Chromatography

9. Gel- Filtration • Method: • Molecules are separated solely on the basis of molecular size • Process can separate molecules ranging in size of MW= 100 to several 1,000,000 Daltons • Popular technique for purifying proteins, nucleic acids, enzymes, etc. • Medium: (Commercial name= Sephadex) • A gel of polysaccharide or other polar polymer • Formulated into small beads with varying degrees of crosslinking within the bead • Results in beads with various pore sizes in the interior of the bead, allowing molecules to separate based on size

10. Gel Filtration Method Solute molecules larger than the pores of the beads can not enter • Purification = to remove any molecules that are not -lactalbumin from a mixture of a protein solution Solute molecules that are small enough to penetrate the beads will be delayed longer Intermediate sized molecules will migrate at a rate in between 1. A stationary phase (beads of gel) consisting of inert material that contains small pores of a fixed size. “sponge” 2. A solution containing solutes of various sizes are applied to a column of the beads at a constant flow rate

11. Gel Filtration Method • Factors that affect how a gel performs and a solute behaves….. • EXCLUSION LIMIT • the molecular mass of the largest molecule that can diffuse through the beads • SEPHADEX G-50 has a limit of 30,000 Daltons • FRACTIONATION RANGE • the range where separation is at a maximum linearity • SEPHADEX G-50 has a range of 1500-30,000 D • BED VOLUME • the final volume taken up by 1 g of dry gel preswollen in water----SEPHADEX G-50 = 9-11 mL/g dry gel

12. Gel Filtration Method • GEL PARTICLE SIZE • size recorded as mesh size • * *how well compounds resolve from a column depend on particle size • 50-100 mesh -larger particles (high flow rates, poor separation) • 400 mesh--superfine particles (poor flow rates, great separation) • 100-200 mesh-- size of SEPHADEX G-50 (Happy Medium) • VOID VOLUME • total space surrounding the gel particles in a packed column • gives an indication as to how long large molecules that will not enter the beads will elute • ELUTION VOLUME • volume of eluting buffer necessary to remove smaller particles that can enter the beads

13. COLUMN:2.5cm X 40cm ELUTING BUFFER: Sephadex is pH stable from 1-10. 0.02M Tris Buffer, pH=7.0 • OPERATING A GEL COLUMN • APPARATUS: SAMPLE VOLUME: (Use 3-4% of bed volume) If too high=poor resolution If too low= eluted fractions too dilute FLOW RATE : too high=reducing interactions of smaller molecules in gel bed too low=eluting all day **optimal flow rate for Sephadex = 0.167-0.333 mL/min ****Column must be packed under continuous flow to provide uniform packing****

14. PROCEDURE • ISOLATION OF MILK WHEY • Complete isolation of milk whey while column is being prepared (see flow chart) • COLUMN PREPARATION • Set up column apparatus (support securely) • Close screw clamp and add 10-15 mL of Tris Buffer • Open screw clamp to allow a slow column flow (0.10mL/min) • Gently pour a well mixed slurry of Sephadex G-50 into column • **the less times you add aliquots of slurry, the better the packing • As the gel settles, continue adding the gel slurry until reaching a bed height of approximately 30cm (Record exact bed height) • Calculate bed volume ( V= π x r2 x h) • NEVER ALLOW THE COLUMN TO RUN DRY!!

15. DETERMINATION OF FRACTIONATION RANGE • When column is completely packed, allow Tris buffer meniscus to drain just before gel bed • Carefully load 2mL of dye mixture (Cobalt Chloride/Blue Dextran) • Allow dye mixture to drain to surface of gel bed • Clamp shut • Carefully add Tris buffer until enough has been added to maintain gel bed surface • Assemble continuous buffer assembly • Allow column to flow at a relatively fast rate • Collect until Blue Dextran completely elutes (Record volume) • This is the VOID VOLUME • Collect until Cobalt Chloride completely elutes (Record volume) • This is the ELUTION VOLUME • Approx. Number of fractions to collect = (ELUTION VOLUME/2mL)

16. PROCEDURE • SAMPLE APPLICATION • After fractionation range has been measured, turn off continuous buffer assembly • Drain buffer just to meniscus of gel bed • Carefully add an appropriate volume of milk whey (a volume that is equivalent to 3-4% of the bed volume) • Allow milk whey to drain just to the surface of the gel bed • Carefully replenish buffer layer above the gel bed • Reconnect continuous buffer assembly • solvent should enter the column at the same rate solvent elutes from the column

17. PROCEDURE • COLUMN DEVELOPMENT& FRACTION ANALYSIS • Allow column to flow at a moderate drop rate • Collect the Void Volume and discard • Immediately begin collecting 2 mL fractions (collected in microcentrifuge tubes) • As fractions are being collected, measure the absorbance at 280nm • Zero the UV-Vis using Tris Buffer at 280 nm • Initially measure the absorbance of every other fraction until protein is detected (positive absorbance) • Continue measuring every fraction thereafter until Absorbance at 280nm reapproaches .000 • SAVE the 2 fractions of the highest absorbance for further characterization next week. • ***Save fractions in freezer. Be sure to label them***

18. SPECTROPHOTOMETRIC CHARACTERIZATION • Rezero the spectrophotometer at 260 nm using Tris buffer • Record the absorbance of 2 most concentrated fractions • Repeat above process at 290 • Record the continuous spectrum between 240-340 nm of two most concentrated fractions • CALCULATIONS • The protein concentration in the isolated fractions can be estimated using the equation below • Protein Conc (mg/mL) = 1.55 A280 - 0.76 A260 • The purity of the protein in the isolated fractions can be estimated one of two ways….. • Calculate the molar absorptivity constant () •  = A280 / (1.00cm) (concentration or protein, g/100mL) • pure lactalbumin has a molar absorptivity of 20.1 • Calculate the ratio of absorbances at 280 and 290 nm • A280 / A290 theoretical ratio of pure lactalbumin is 1.30 • Compare continuous spectrum of purified fractions to standard Lactalbumin

19. Isolation and Characterization of alpha-Lactalbumin Experiment#4 Week#2--Protein purification/characterization using Electrophoresis/ Bradford Assay

20. Protein Characterization • Purpose: • To assess the purity of the purified whey by electrophoresis • To estimate the concentration of isolated protein using Bradford Assay

21. Gel Electrophoresis • What is electrophoresis? “Electro”=the energy of electricity “phoros”= to carry across • The movement of charged molecules in an electric field • Degree of migration through electric field depends on the size, shape, charge, and chemical makeup • Movement is a function of • the net charge on a molecule • the retarding frictional forces as the molecule moves through the experimental media

22. Usefulness of Electrophoresis • Electrophoretic results can be a useful tool • PURITY • if well displays only one band after staining (electrophoretically pure) • if 2 or more bands are visible sample contains multicomponents (impure) • IDENTITY • when compared alongside standards of known identity, one can compare the migration distances of both the known and unknown • CONTINUAL USE • individual bands (representing separated compounds) can be removed and subjected to further analysis

23. Gel Electrophoresis = technique of forcing molecules across a span of gel • Medium : • Polyacrylamide Gel (PAGE) • Sodium Dodecyl Sulfate Polyacrylamide (SDS-PAGE) **used primarily for proteins • Agarose (used frequently for DNA) • Polyacrylamide Gels • Cross-linked polymer of acrylamide and methylene-bis acrylamide • Have high resolution for small to intermediate sized molecules • acceptance of relatively large sample sizes • physical stability

24. Separation of Molecules in PAGE • Separation is due to molecules passing through a continuous pore environment • NO VOID VOLUME! • Resolution can be improved by varying the gel composition throughout a single gel (GRADIENT GEL) • As acrylamide concentration decreases • gels give larger pores allowing analysis of larger molecules • As acrylamide concentration increases • gels give smaller pores allowing analysis of smaller molecules • Proteins with molecular weights ranging from 10,000-100,000 can be separated on gels with 11% acrylamide • small molecules > intermediate molecules > larger molecules

25. Comparison of Techniques

26. SDS-PAGE • SDS = Sodium Dodecyl Sulfate • An anionic detergent which denatures proteins by “wrapping around” the polypeptide backbone • Confers a negative charge to the polypeptide in proportion to its length • Disulfide bridges are further reduced so that the protein will adopt a random coil configuration • usually done with 2-mercaptoethanol • Migration is now solely based on molecular weight

28. PAGE electrophoresis for proteins is done in vertical slab arrangement • Apparatus:

29. Electrophoresis Method • Visualization • Since proteins can’t be seen on the gel, they must be made visible by staining • The entire gel is stained with a dye such as Coomassie Brilliant Blue that will bind to the proteins • The gel is destained to remove any unbound dye (proteins then appear as discrete bands in the gel) • Determination of Molecular Weight • Marker proteins of known molecular weight are electrophoresed alongside the protein samples • Relative mobilities of known MW markers are compared to the relative mobility of the unknown protein • Plot of logMW vs the mobilities is made and the relative mobility of the unknown is used to determine its MW

30. Electrophoresisresults Relative Mobility= distance traveled by protein band distance traveled by gel front

31. Protein Quantitation • Several methods to choose from (method differences based on sensitivity and interferences) • Biuret assay • Lowry assay • BCA protein assay • Bradford assay • All methods adhere to Beer’s Law • Series of protein standards are assayed at the analytical wavelength • Absorbances of unknowns are used to interpolate concentration • ** all unknowns should be in the range of the standards

32. Bradford Assay • Method • can be used to determine proteins in the range of 1 to 20ug • is very rapid and has very few interferences by nonprotein components • based on protein binding of a dye (thought to be to arginine residues) • The binding of Coomassie Brilliant Blue dye to protein in acidic solution shifts the wavelength of absorbance from 465nm to 595nm • Absorbance of 595 nm is directly related to the concentration of the protein • Calibration curve is prepared using bovine serum albumin (BSA) as a standard (Or bovinegamma-globulin)

33. Procedure • Complete UV spectrophotometric measurements/scans if necessary • Electrophoresis • Fill inner electrophoresis chamber with running buffer (Tris-SDS-Glycine) • Carefully and properly insert PAGE precast gel cassettes into chamber • Fill outer electrophoresis chamber to fill line with running buffer • Prepare protein samples • (Crude Whey, 2 Fractions saved from last week) • Mix 100uL of protein samples with 100uL of sample application buffer • Add 8uL of 25% SDS • Add 6uL of mercaptoethanol • Prepare MW marker standard • 20 uL MW marker + 20uL sample application buffer • Add 2uL SDS and 1uL mercaptoethanol

34. Procedure (continued) • Electrophoresis (continued) • Prepare protein samples (continued) • Heat protein mixtures in a boiling water bath for 3 minutes to denature the proteins • Cool to room temperature • Inject 10 uL of each cooled sample into a well on the PAGE gel • Inject 10uL of MW protein marker solution in at least one lane • (Record placement of samples in your notebook) • Connect appropriately to power supply and run until the tracking dye is at the bottom of the gel • The current should be set at 50 mA

35. Procedure • Electrophoresis (continued) • Disconnect power supply when the tracking dye almost reaches the bottom of the gel and carefully separate the gel from the glass plates • Carefully transfer the gel to a tray containing Coomassie Blue dye staining solution (soak the gel for 30 minutes—or overnight) • Decant the dye solution and add de-staining solution (65% water: 30% methanol: 5% acetic acid solution) • Continue destaining and periodically replace the destaining solution with fresh solution until the background color is removed (may take 24 hours or so) • View the de-stained gel under a light source • Draw a picture of the gel in your notebook • Calculate Relative Mobility for all samples

36. Procedure • Bradford Assay • Formation of Standard Curve: • Standard gamma globulin = 0.1mg/mL • To each of the following sample mixtures, add 5.0 mL Bradford dye reagent , wait for 5 minutes and record the absorbance at 595nm (adjust protein concentrations as necessary to keep all absorbances between .05-1.000) • Reference--BLANK (0mg): 1.0mL DI water • Zero spectrophotometer at 595nm • Standard #1 = 0.1mL gamma globulin std + 0.9mL DI • Standard #2 = 0.2 mL gamma globulin std + 0.8mL DI • Standard #3 = 0.4 mL gamma globulin std + 0.6 mL DI • Standard #4 = 0.6 mL gamma globulin std + 0.4 mL DI • Standard #5 = 0.8 mL gamma globulin std + 0.2 mL DI • Calculate the total mg of protein • Plot total mg protein vs. Absorbance at 595nm • Determine equation of line: y = mx + b

37. Procedure • Bradford Assay (continued) • Analysis of unknown protein samples • Choose appropriate volumes(by trial and error) for each of the protein samples (Crude Whey, 2 fractions saved from last week), add enough DI water to give 1.0mL of diluted protein add 5 mL of Bradford dye, wait for 5 minutes and record absorbance at 595 nm • Make sure all sample absorbances are in the range of the standard curve • PROTEIN SAMPLES • Crude Whey#1 = (recommend 0.05mL-0.10mL) • Gel Filtration Fractions= (recommend 0.3-0.6mL) • Using the line equation from the standard curve, calculate relative concentration (reported as total mg of protein) in each of the samples of fraction and crude whey

38. Data Analysis • Electrophoresis • Compare relative mobilities of standard lactalbumin to purified fraction to crude whey • Estimate MW of purified fraction by comparing the relative mobility of purified fraction to a plot of (log MW vs relative mobility) of standard biomarker • Comment on the efficiency of the gel filtration in purifying your sample • Is the isolated fraction lactalbumin? • Is lactalbumin the predominant protein? • Bradford Assay • Construct Beer’s Law plot using standard concentrations and absorbances • Calculate the relative protein concentration of the crude whey, and purified fractions