Transcript Protein Separation

Isolation and Characterization of
alpha-Lactalbumin
Experiment#4
Week#1--Protein purification using
Gel Filtration
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
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
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)


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
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)
Sediment (white)
Supernatant
Removes caseins and
phosphoproteins
Change pH to 4.6 (using HCl)
Discard
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
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)

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
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
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
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
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

OPERATING A GEL COLUMN
• 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
****Column must be packed
too low=eluting all day
under continuous flow to provide
**optimal flow rate for
uniform packing****
Sephadex = 0.167-0.333 mL/min
PROCEDURE

ISOLATION OF MILK WHEY
• Complete isolation of milk whey while column is being prepared
(see flow chart)

COLUMN PREPARATION
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•
•
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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!!

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)
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
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***

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
Isolation and Characterization of
alpha-Lactalbumin
Experiment#4
Week#2--Protein
purification/characterization using
Electrophoresis/ Bradford Assay
Protein Characterization

Purpose:
• To assess the purity of the purified whey by
electrophoresis
• To estimate the concentration of isolated
protein using Bradford Assay
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
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

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
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
Comparison of Techniques
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

PAGE electrophoresis for proteins is done
in vertical slab arrangement
• Apparatus:
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
Electrophoresis results
Relative Mobility= distance traveled by protein band
distance traveled by gel front
Protein Quantitation

Several methods to choose from (method
differences based on sensitivity and interferences)
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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
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)
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
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
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
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
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
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