2. Know the Characteristics of Your ProteinKnow the Characteristics of Your Protein
Ex. Human Serum AlbuminEx. Human Serum Albumin
Sequence of AminoSequence of Amino
AcidsAcids
MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGEMKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE
EHFKGLVLIA FSQYLQQCPFDEHVKLVNEL TEFAKTCVADEHFKGLVLIA FSQYLQQCPFDEHVKLVNEL TEFAKTCVAD
ESHAGCEKSL HTLFGDELCK VASLRETYGMADCCEKQEPESHAGCEKSL HTLFGDELCK VASLRETYGMADCCEKQEP
ERNECFLSHK DDSPDLPKLK PDPNTLCDEFKADEKKFWGKERNECFLSHK DDSPDLPKLK PDPNTLCDEFKADEKKFWGK
YLYEIARRHP YFYAPELLYYANKYNGVFQE CCQAEDKGACYLYEIARRHP YFYAPELLYYANKYNGVFQE CCQAEDKGAC
LLPKIETMRE KVLTSSARQR LRCASIQKFG ERALKAWSVALLPKIETMRE KVLTSSARQR LRCASIQKFG ERALKAWSVA
RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDRRLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR
ADLAKYICDN QDTISSKLKECCDKPLLEKS HCIAEVEKDAADLAKYICDN QDTISSKLKECCDKPLLEKS HCIAEVEKDA
IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRRIPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR
HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKLHPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL
KHLVDEPQNL IKQNCDQFEKLGEYGFQNAL IVRYTRKVPQKHLVDEPQNL IKQNCDQFEKLGEYGFQNAL IVRYTRKVPQ
VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLILVSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL
NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETYNRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY
VPKAFDEKLF TFHADICTLPDTEKQIKKQT ALVELLKHKPVPKAFDEKLF TFHADICTLPDTEKQIKKQT ALVELLKHKP
KATEEQLKTV MENFVAFVDK CCAADDKEACFAVEGPKLVKATEEQLKTV MENFVAFVDK CCAADDKEACFAVEGPKLV
WSTQTALAWSTQTALA
Tertiary StructureTertiary Structure
3. Know the Characteristics of YourKnow the Characteristics of Your
ProteinProtein
Human Serum Albumin:Human Serum Albumin:
MW (molecular weight = 69,000MW (molecular weight = 69,000
Daltons (69 kD)Daltons (69 kD)
pI (isoelectric point) = 5.82pI (isoelectric point) = 5.82
Hydropathicity (=hydrophobicity) =Hydropathicity (=hydrophobicity) =
-.395-.395
4. LARGE SCALE PROTEIN PRODUCTION
Transfected cells grown to confluence in
10 x T175 flasks
Wash with sterile PBS to remove contaminant
proteins from serum (BSA)
Culture cells in serum free medium (growth arrest)
3 x medium exchange every 48/76 hours
CONDITIONED MEDIUM READY FOR PURIFICATION
5. EASY 2 STEPS PROTEIN PURIFICATION
AFFINITY CHROMATOGRAPHY
GEL FILTRATION
0
500
Absorptionat280nm(mAU)
1000
1500
2000
2500
500 mM Imidazole
-45kDa
Elution volume (ml)
Vo 10 15 20 25
0
500
1000
1500
Absorptionat280nm(mAU)
2000
-45kDa
6. GLYCOSYLATIONGLYCOSYLATION
– Mammalian sugar chains have highlyMammalian sugar chains have highly
complex structurescomplex structures
– Good for functional studiesGood for functional studies
– Big problem for protein crystallizationBig problem for protein crystallization
SOLUTIONSSOLUTIONS
– Mutagenesis of glycosylation sitesMutagenesis of glycosylation sites
– Enzymatic deglycosylationEnzymatic deglycosylation
– Engineered cell lines (CHO Lec strains)Engineered cell lines (CHO Lec strains)
– Chemical inhibitors of glycosylationChemical inhibitors of glycosylation
pathwaypathway
– Insect cells (simple sugars)Insect cells (simple sugars)
8. Typical Protein Production Process FlowTypical Protein Production Process Flow
(Feed 2)
(Feed 3)
(Feed 4)
Chrom 1
Chrom 3
Cryo-preservation
Concentration /
Diafiltration
Centrifuge
Viral Removal
Filtration
(Feed1)Inoculum Expansion
(Spinner Bottles)
Ampule Thaw
Chrom 2
9. Media Prep
Media Prep
Working Cell
Bank
Working Cell
Bank
Sub-
Culture
Sub-
Culture
Inoculum
Sub-
Culture
Sub-
Culture Sub-
Culture
Sub-
Culture Sub-
Culture
Sub-
Culture Sub-
Culture
Sub-
Culture
Large Scale Bioreactor
Wave
Bag
Wave
Bag
Seed Bioreactors
Fermentation
150L
Bioreactor
750L
Bioreactor
5,000L
Bioreactor
26,000L
Bioreactor
Depth
Filtration
Depth
Filtration
Collection
Collection
Centrifuge
Centrifuge
Harvest/Recovery
Harvest
Collection
Tank
1,500L
Harvest
Collection
Tank
1,500L
Filter
Chromatography
Skid
Anion Exchange
Chromatography (QXL)
Column
Eluate
Hold
Tank
8,000L
Eluate
Hold
Tank
8,000L
Eluate
Hold
Tank
6,000L
Eluate
Hold
Tank
6,000L
Filter
Chromatography
Skid
Protein A
Chromatography
Column
Chromatography
Skid
Column
Eluate
Hold
Tank
20,000L
Eluate
Hold
Tank
20,000L
Hydrophobic Interaction
Chromatography (HIC)
Eluate
Hold
Tank
20,000L
Eluate
Hold
Tank
20,000L
Viral
Inactivation
Eluate
Hold
Tank
5,000L
Eluate
Hold
Tank
5,000L
Filter
Chromatography
Skid
Anion Exchange
Chromatography
(QFF - Fast Flow)
Column
Post-viral
Hold
Vessel
3,000L
Post-viral
Hold
Vessel
3,000L
Viral Filtering Ultra Filtration
Diafiltration
Bulk
Fill
Purification
24 days 31 days
8 days
1 day
Mfg Process OverviewMfg Process Overview
10.
11. cGMP Pilot Plant Manufacturing FacilitycGMP Pilot Plant Manufacturing Facility
Small
Scale
Manufact.
DSP 1 DSP 2 DSP 3 DSP 4
Small
Scale
Manufact.
Medium
Scale
Manufact.
Large
Scale
Manufacturing
Media/
Buffer Prep.
Equipment
Wash
Inoc.
Break
Room
Toilets/
Lockers
Toilets/
Lockers
Toilet
Toilet
Toilets/
Lockers
Toilets/
Lockers
Return
Clean
Street / Plant
Employee Entrance
Visitors/
Admin.
Entrance
Waste Dock Shipping &
Receiving
Lab Dock
Support
Lobby
Support
QC Lab
Future Expansion
Warehouse
Dispensary
Warehouse
Shipping &
Receiving
Offices Building
Utilities
Maintenance
Waste
Staging
Cylinder/Solvent
Staging
Filling Suite
Waste
Stage Process Utilities
12. Clarification orClarification or
Removal of Cells andRemoval of Cells and
Cell DebrisCell Debris
Using CentrifugationUsing Centrifugation
(Using Depth Filtration)(Using Depth Filtration)
14. Separation of particles from liquid by
applying
a pressure to the solution to force the
solution through a filter. Filters are materials
with pores.
Particles larger than the pore size of the
filter are retained by the filter.
Particles smaller than the pore size of the
filter pass through the filter along with the
FiltrationFiltration
15. Uses cross flow to reduce build up
of retained components on the
membrane surface
Allows filtration of high fouling
streams and high resolution
Tangential Flow FiltrationTangential Flow Filtration
16. Tangential Flow Filtration – TFFTangential Flow Filtration – TFF
Separation of Protein of InterestSeparation of Protein of Interest
Using TFF with the right cut off filters, the protein ofUsing TFF with the right cut off filters, the protein of
interest can be separated from other proteins andinterest can be separated from other proteins and
molecules in the clarified medium.molecules in the clarified medium.
HSAHSA has a molecular weight of 69KD. To make surehas a molecular weight of 69KD. To make sure
that the protein of interest is retained, a 10KD cut-that the protein of interest is retained, a 10KD cut-
off filter is used.off filter is used.
After we concentrate or ultrafilter our protein, we canAfter we concentrate or ultrafilter our protein, we can
diafilter, adding the phosphate buffer at pH 7.1diafilter, adding the phosphate buffer at pH 7.1
that we will use to equilibrate our affinity columnthat we will use to equilibrate our affinity column
to prepare for affinity chromatography ofto prepare for affinity chromatography of HSAHSA..
17. Overview of TFF SOPOverview of TFF SOP
Prepare buffer:Prepare buffer:
Sodium phosphate buffer pH 7.1Sodium phosphate buffer pH 7.1
Set up the apparatus-Set up the apparatus-CAUTION Stored in NaOHCAUTION Stored in NaOH
Flush with water-Flush with water-CAUTION Stored in NaOHCAUTION Stored in NaOH
Adjust flow rate to 30-50ml/minAdjust flow rate to 30-50ml/min
Flush retentate lineFlush retentate line
Flush permeate lineFlush permeate line
Precondition with buffer (just the permeate line)Precondition with buffer (just the permeate line)
Perform TFFPerform TFF
Prepare cleaning solution (NaOH)Prepare cleaning solution (NaOH)
Flush with waterFlush with water
Flush with NaOH to clean and storeFlush with NaOH to clean and store
20. How TFF Concentrates andHow TFF Concentrates and
DiafiltersDiafilters
the Protein of Interestthe Protein of Interest
21. Low PressureLow Pressure
ProductionProduction
ChromatographyChromatography
The System: Components andThe System: Components and
ProcessesProcesses
The Media: Affinity, IonThe Media: Affinity, Ion
Exchange, HydrophobicExchange, Hydrophobic
Interaction ChromatographyInteraction Chromatography
and Gel Filtrationand Gel Filtration
22. 22
Sample
Separation
technique
FractionationFractionation
Purification is a Multi-Step Procedure.
Is there activity?Set aside
NN
oo
Combine
Fractionsyesyes
Monitor purityMonitor purity
Assay total protein
Assay enzyme activity
Pure?
Prepare for analytical technique
yesyes
NN
oo
Repeat with anotherRepeat with another
separationseparation
technique until puretechnique until pure
23. 23
General Protein PurificationGeneral Protein Purification
SchemeScheme
• Grow cells in
media
(vector+tag)
•Bacteria
Suspension
•Bioreactor
Purification Strategy
Expression
SDS PAGE Assay
Solubility
Aggregation
Recombination
Characterization
Mass Spectroscopy
X-ray Crystallography
Functional Assay
Lyse the cells
(appropriate
buffer)
Centrifuge
Collect the pellet
24. 24
1. Evaluate an assay for the protein of interest
2. Shortlist a method to have a reasonable source for that activity
Set Protein Purification Strategy
25. 25
Preparing the samplePreparing the sample
(Crude Extract)(Crude Extract)
Protein from cells or tissueProtein from cells or tissue
Microbial cellsMicrobial cells
or tissueor tissue
Break cells,Break cells,
Blender,Blender,
homogenizer,homogenizer,
sonication,sonication,
pressurepressure
osmoticosmotic Pellet with intactPellet with intact
cells, organelles,cells, organelles,
membranes andmembranes and
membrane proteinsmembrane proteins
Supernatant withSupernatant with
Soluble proteinSoluble protein
26. 26
As the column separates the proteins inAs the column separates the proteins in
the mixture, the “the mixture, the “ effluenteffluent ” drips into a” drips into a
series of fraction tubes that are movingseries of fraction tubes that are moving
at a specific rate of speed. These tubesat a specific rate of speed. These tubes
are calledare called fractionsfractions..
Here we are showing 20 tubes. FractionHere we are showing 20 tubes. Fraction
collectors in most labs have about 75-collectors in most labs have about 75-
200 tubes.200 tubes.
How do we know which fractions containHow do we know which fractions contain
protein? Total protein a can be estimatedprotein? Total protein a can be estimated
by taking the absorbance at 280 nm in aby taking the absorbance at 280 nm in a
spectrophotometer. Aromatic aminospectrophotometer. Aromatic amino
acids absorb light at this wavelengthacids absorb light at this wavelength
causing all proteins to have absorbancecausing all proteins to have absorbance
at 280nm. Many fraction collectorsat 280nm. Many fraction collectors
measure the A280 as the column ismeasure the A280 as the column is
running.running.
Collect fractions.Collect fractions.
27. 27
A280
Plot valuesPlot values
00 00 00 22 55 22 00 00 00 22 55 88 55 22 00 00 22 55 22 00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction #
Question 1. How do we knowQuestion 1. How do we know
which fractions contain protein?which fractions contain protein?
28. 28
• Total protein a can be estimated by
taking the absorbance at 280 nm in
a spectrophotometer.
• The values can be plotted against
the fraction number in is what is
called an elution profile.
• Notice the peaks on the graph.
These indicate where the fractions
are that contain protein.
Question 1. How do we know
which fractions contain protein?
A280
00 00 00 22 55 22 00 00 00 22 55 88 55 22 00 00 22 55 22
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Fraction
#
A280
Fraction #
PeaksPeaks
29. 29
• Enzyme activity can be
determined by performing an
enzyme assay on each fraction
that contains protein.
Which fractions contained the desired protein?
A280
00 00 00 22 55 22 00 00 00 22 55 88 55 22 00 00 22 55 22 00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
#
A280
Fraction #
Enz. Assay.Enz. Assay.Enz. Assay.Enz. Assay.
Fraction
#
30. 30
• Enzyme activity can beEnzyme activity can be
determined by performing andetermined by performing an
enzyme assay on each fractionenzyme assay on each fraction
that contains protein.that contains protein.
• Notice the results of the enzymeNotice the results of the enzyme
assay. The highest activityassay. The highest activity
corresponds to one of the peaks.corresponds to one of the peaks.
• Now we can have them discardNow we can have them discard
tubes that don’t have enzymetubes that don’t have enzyme
activity.activity.
Which fractions contained the desired enzyme?
A280
00 00 00 22 55 22 00 00 00 22 55 88 55 22 00 00 22 55 22 00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
#
A280
Fraction #
EnzAssay
Results
32. Peristaltic PumpPeristaltic Pump
Creates a gentleCreates a gentle
squeezing action tosqueezing action to
move fluid throughmove fluid through
flexible tubing.flexible tubing.
33. 33
The Way to ChromatographyThe Way to Chromatography
In order to isolate sufficient quantities ofIn order to isolate sufficient quantities of
protein, you may need to start with kilogramprotein, you may need to start with kilogram
quantities of source (i.e. bacteria, tissues,quantities of source (i.e. bacteria, tissues,
etc.) These amounts can best be handledetc.) These amounts can best be handled
using precipitation methods (e.g.using precipitation methods (e.g.
ammonium sulfate precipitation). Later inammonium sulfate precipitation). Later in
the purification, large columns can be usedthe purification, large columns can be used
to handle gram to milligram quantities.to handle gram to milligram quantities.
Amounts handled on gels are typically inAmounts handled on gels are typically in
microgram quantities.microgram quantities.
34. Liquid Column ChromatographyLiquid Column Chromatography
ProcessProcess
Purge Air from System with Equilibration BufferPurge Air from System with Equilibration Buffer
Pack Column with Beads (e.g. ion exchange, HIC,Pack Column with Beads (e.g. ion exchange, HIC,
affinity or gel filtration beads)affinity or gel filtration beads)
Equilibrate Column with Equilibration BufferEquilibrate Column with Equilibration Buffer
Load Column with Filtrate containing Protein ofLoad Column with Filtrate containing Protein of
Interest in Equilibration BufferInterest in Equilibration Buffer
Wash Column with Equilibration BufferWash Column with Equilibration Buffer
Elute Protein of Interest with Elution Buffer of HighElute Protein of Interest with Elution Buffer of High
or Low Salt or pHor Low Salt or pH
Regenerate Column or Clean and StoreRegenerate Column or Clean and Store
35. LP LC ComponentsLP LC Components
Mixer for Buffers, Filtrate with Protein ofMixer for Buffers, Filtrate with Protein of
Interest, Cleaning SolutionsInterest, Cleaning Solutions
Peristaltic PumpPeristaltic Pump
Injector to Inject Small Sample (in ourInjector to Inject Small Sample (in our
case for HETP Analysis)case for HETP Analysis)
Chromatography Column and MediaChromatography Column and Media
(Beads)(Beads)
Conductivity MeterConductivity Meter
UV DetectorUV Detector
36. UV DetectorUV Detector
Detects proteins coming out of theDetects proteins coming out of the
column by measuring absorbancecolumn by measuring absorbance
at 280nmat 280nm
37. Conductivity MeterConductivity Meter
Measures the amount of salt in theMeasures the amount of salt in the
buffers – high salt or low salt arebuffers – high salt or low salt are
often used to elute the protein ofoften used to elute the protein of
interest from the chromatographyinterest from the chromatography
beads.beads.
Also measures the bolus of salt thatAlso measures the bolus of salt that
may be used to determine themay be used to determine the
efficiency of column packing (HETP)efficiency of column packing (HETP)
38. 38
PropertyProperty MethodsMethods
SolubilitySolubility PrecipitationPrecipitation
with ammoniumwith ammonium
sulfate (saltingsulfate (salting
out)*out)*
Size / shapeSize / shape Size-exclusionSize-exclusion
chromotographychromotography
IsoelectricpoIsoelectricpo
int (charge)int (charge)
Ion exhangeIon exhange
chromatographychromatography
binding tobinding to
smallsmall
moleculesmolecules
AffinityAffinity
chromatographychromatography
Common methods of protein purification
*Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes.
It is commonly one of the first steps in a purification scheme.
Purification procedures attempt toPurification procedures attempt to
maintain the protein in native form.maintain the protein in native form.
Although some proteins can beAlthough some proteins can be
re-natured, most cannot!re-natured, most cannot!
To purify a protein from a mixture,To purify a protein from a mixture,
biochemists exploit the ways thatbiochemists exploit the ways that
individual proteins differ from oneindividual proteins differ from one
another. They differ in:another. They differ in:
Thermal stabilityThermal stability: For most protein: For most protein
purifications, all steps are carriedpurifications, all steps are carried
out at ~5°C to slow downout at ~5°C to slow down
degradation processes.degradation processes.
39. 39
Picture of protein gelPicture of protein gel
with lanes showingwith lanes showing
sequential purificationsequential purification
stepssteps
ProcedProced
ureure
FractioFractio
n voln vol
(ml)(ml)
TotalTotal
ProtProt
(mg)(mg)
ActivityActivity
(units)(units)
SpecificSpecific
activityactivity
Units/Units/
mgmg
CrudeCrude
cellularcellular
extractextract
14001400 1000010000 100,000100,000 1010
Size-Size-
exclusioexclusio
nn
9090 400400 80,00080,000 200200
IonIon
exchangexchang
ee
8080 100100 60,00060,000 600600
Note: The type and order of steps are customized for each protein to be
purified. An effective purification step results in a high yield (minimal loss
of enzyme activity) and large purification factor (large increase in specific
activity).
Purification YieldPurification Yield
40. 40
Chromatographic ModeChromatographic Mode AcronymAcronym Separation PrincipleSeparation Principle
Non-interactive modes of liquid chromatographyNon-interactive modes of liquid chromatography
Size-exclusion chromatographySize-exclusion chromatography SECSEC Differences in molecular sizeDifferences in molecular size
Agarose chromatography (forAgarose chromatography (for
DNA) for DNA binding proteinsDNA) for DNA binding proteins -- Diff. in length and flexibilityDiff. in length and flexibility
Interactive modes of liquid chromatographyInteractive modes of liquid chromatography
Ion-exchange chromatographyIon-exchange chromatography IECIEC Electrostatic interactionsElectrostatic interactions
Normal-phase chromatographyNormal-phase chromatography NPCNPC Polar interactionsPolar interactions
Reversed-phase chromtographyReversed-phase chromtography RPCRPC Dispersive interactions*Dispersive interactions*
Hydrophobic interactionHydrophobic interaction
chromatographychromatography HICHIC Dispersive interactions*Dispersive interactions*
Affinity chromatographyAffinity chromatography ACAC Biospecific interactionBiospecific interaction
Metal interactionMetal interaction
chromatographychromatography MICMIC Complex w/ an immobilizedComplex w/ an immobilized
metalmetal
Chromatographic Modes of Protein Purification
* Induced dipole – induced dipole interactions
44. 44
Affinity Chromatography
Binding Capacity (mg/ml) medium
12mg of histag proteins (MW= 27kDa)
Depends on Molecular weight
Degree of substitution /ml medium
~15mmol Ni2+
Backpressure ~43psi
Change the guard column filter
45. 45
Biopolymer (phenyl agarose - Binding Surface)
Driving force for hydrophobic adsorption
Water molecules surround the analyte and the
binding surface.
When a hydrophobic region of a biopolymer binds to
the surface of a mildly hydrophobic stationary
phase, hydrophilic water molecules are effectively
released from the surrounding hydrophobic areas
causing a thermodynamically favorable change in
entropy.
Temperature plays a strong role
Ammonium sulfate, by virtue of its good
salting-out properties and high solubility in water is
used as an eluting buffer
Hydrophobic Interaction Chromatography
Hydrophobic region
46. 46
ION –EXCHANGE 1ION –EXCHANGE 1
First, to determine theFirst, to determine the
charge on a protein, given itscharge on a protein, given its
pI and the pH.pI and the pH.
Ion-exchange columnIon-exchange column
chromatography separateschromatography separates
proteins on the basis ofproteins on the basis of
charge.charge.
We will start with 4 proteins.We will start with 4 proteins.
pH 7.2pH 7.2
Positive charged columnPositive charged column
60 Kd
Low pI (6)
20 Kd
Low pI (7)
20 Kd
Medium pI (7)
5 Kd
Hi pI (8)
47. 47
pos
• The matrix of an ion exchange
is positively charged.
• What do you think will
happen?pos
pos
pos
pos
pos
pos
Run columnRun column
pos
pos
pos
pos
pos
pos
48. 48
• The matrix of an ion exchange
is positively charged.
• Only the pos charged proteins
run through the pos charged
column. The others “stick” to
the column.
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
pos
49. 49
Fractogel matrix is a methacrylate resin upon which polyelectrolyte
Chains (or tentacles) have been grafted. (Novagen)
Ion Exchange Chromatography
Globular
Protein
Deformation due to interaction with
conventional ion exchanger
Maintenance of conformation
while interacting with tentacle
ion exchanger
51. 51
Gel filtration columnGel filtration column
chromatography separateschromatography separates
proteins on the basis of size.proteins on the basis of size.
We will start with 4 proteins.We will start with 4 proteins.
You will want to purify theYou will want to purify the
“yellow one”“yellow one”
60 Kd
Low pI (6)
20 Kd
Low pI (7)
20 Kd
Medium pI (7)
5 Kd
Hi pI (8)
Gel Filtration
52. 52
The matrix of a size-exclusionThe matrix of a size-exclusion
chromatography column ischromatography column is
porous beads.porous beads.
Run columnRun column
53. 53
The matrix of a gel filtrationThe matrix of a gel filtration
column are beads withcolumn are beads with
pores.pores.
The largeThe large graygray proteinsproteins
can’t fit in pores so flowscan’t fit in pores so flows
faster.faster.
TheThe redred // yellowyellow mediummedium
sized proteins get trapped insized proteins get trapped in
the pores.the pores.
TheThe blackblack small proteinssmall proteins
stay trapped in pores longer.stay trapped in pores longer.
55. 55
ATP immobilized on polyacrylamide resin
DNA Binding Proteins
Heparin Sepharose
Negatively charged proteins (pI >7) are not captured/separated effectively.
56. 56
Capillary Electrochromatography
• CEC is an electrokinetic separation technique
• Fused-silica capillaries packed with stationary phase
• Separation based on electro-osmotically driven flow
• Higher selectivity due to the combination of chromatography and
electrophoresis
Fused silica tube filled with porous methacrylamide-stearyl
methacrylate-dimethyldiallyl ammonium chloride monolithic
polymers, 80 x 0.5mm i.d., 5.5kV. High Plate count ~ 400,000
Height Equivalent to a Theoretical Plate /Plate Count (HETP) H = L/N
number of plates N = 16(t/W)2
where L = column length, t = retention time, and W = peak width at baseline
61. 61
REFERENCESREFERENCES
Christian G. Huber, Biopolymer Chromatography, EncylcopediaChristian G. Huber, Biopolymer Chromatography, Encylcopedia
in analytical chemistry, 2000in analytical chemistry, 2000
www.qiagen.comwww.qiagen.com
www.novagen.comwww.novagen.com
http://lsvl.la.asu.edu/resources/mamajis/chromatography/chromhttp://lsvl.la.asu.edu/resources/mamajis/chromatography/chrom
atography.htmlatography.html
http://www.cellmigration.org/resource/discovery/discovery_protehttp://www.cellmigration.org/resource/discovery/discovery_prote
omics_approaches.htmlomics_approaches.html
http://www.capital-hplc.co.ukhttp://www.capital-hplc.co.uk
http://www.ls.huji.ac.il/~purificationhttp://www.ls.huji.ac.il/~purification
www.biovectra.comwww.biovectra.com
http://www.ls.huji.ac.il/~purificationhttp://www.ls.huji.ac.il/~purification
Notas del editor
Continuous multichamber disc-stack centrifuge. The bowl contains a
number of parallel discs providing a large clarifying surface with a small sedimentation distance. The sludge (cells) is removed through
This flow chart is more simplified than the other one. What do you think?
For “separation graphic” show image of the chromatographic apparatus instead of a box.
Combine fractions box: Graphic of little tubes pouring into a bigger beaker.
We need to fit in a short text block with a description of the steps in order using numbers to label the steps.
ANIMATION.
Show cartoon of microbial cells or tissue.
Break the cells
Have colors which show cellular membranes and junk contrasting with the proteins into solution.
User clicks the PLOT VALUES button.
Animation: A graph grows.
After the animation ends
The next appears and the user is prompted to discard the fractions that don’t belong by clickin on the tubes.
Go to next screen to see what it might look like.
Should the A280 value table go away? And just leave the profile to “declutter” the screen.
Peter feels strongly that they should not be encouraged to throw away at this point. Perhaps set aside would be a better term, or eliminate altogether.
they are prompted to perfom an enzyme activity assay on the remaining tubes.
They are prompted to click the “Enzyme Assay” button.
Go to next screen to see results.
Show the color of activity tapers off in intensity.
Go to next screen.
Since so few assays use color, Peter would like these to be absorbance values.
Creates a gentle squeezing action to move fluid through flexible tubing.
In this example three rollers on rotating arms pinch the tube against an arc and move the fluid along. There are usually three or four sets of rollers
Should these link to a Glossary? I don’t think so.
The student should not worry if they don’t understand the techniques at this point. This is just to introduce some terminology in context.
Is the table a good idea? If so, it needs to be more complete.
JB Table is a good idea - we don’t want the students to consider SDS PAGE to be a purification technique, though, so I removed it from the table.
More background content needed? Theory of it?
We need the info on the proteins.
Should the proteins all be the same size?
Students click on “run column”
Go to next slide to see results.
Just a next button?
I might have gotten the charges mixed up. Let’s make sure we get this straight.
We might need a legend (or something of the kind) on every page???
Do we need more content on what is size? How is it measure? MW.
Pick real numbers is Kd correct?
Show them all repeated together.
Should these proteins be more blob like than perfect circles?
Each color represents a different size/charge.
Perhaps we should develop a little “protein” profile next to each blob.
Protein 1.
60 kD
I think that we should make it so that we make one of the proteins the one that the student wants to purify.
In both Gel filtration and Ion exchange, let’s make it so that neither method works.
The final point we can make is that you have to do both separation methods in sequence.