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 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. PROTEIN PURIFICATION TECHNIQUES
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: Medium: (Commercial name= Sephadex)
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
Purification = to remove any molecules that are not
 -lactalbumin from a mixture of a protein solution
Solute molecules larger than the pores of the beads can not enter
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 ,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 VOID VOLUME ELUTION VOLUME
size recorded as mesh size
* *how well compounds resolve from a column depend on particle size
mesh -larger particles (high flow rates, poor separation)
400 mesh--superfine particles (poor flow rates, great separation)
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. OPERATING A GEL COLUMN COLUMN: 2.5cm X 40cm
APPARATUS:
COLUMN: 2.5cm X 40cm
ELUTING BUFFER: Sephadex is pH stable from 1-10.
0.02M Tris Buffer, pH=7.0
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 = mL/min
****Column must be packed under continuous flow to provide uniform packing****

14. PROCEDURE ISOLATION OF MILK WHEY COLUMN PREPARATION
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 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 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 A 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 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. Electrophoresis results
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 bovine gamma-globulin)

33. Procedure Electrophoresis
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 )
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 mL DI
Standard #4 = 0.6 mL gamma globulin std mL DI
Standard #5 = 0.8 mL gamma globulin std 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 mL)
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 Bradford Assay
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