6Main PPT Protein Purification
Transcript of 6Main PPT Protein Purification
Starting Sample
DepleteFractionate
PurifyConcentrate
Desalt/ Remove
DetergentAnalyze
Simplifying Sample Prep
Novel methods for Protein Purification using Pall Life Sciences Chromatography resins.
Monica IsaacsTechnical Marketing Manager
Pall Biosciences - ASIA
Agenda
� Recommended strategies for Sample Preparation� Chromatography Methods
� Special feature resins� Platforms for Chromatography� Ultrafiltration� Western Blotting� Protecting your column and samples� Summary� Questions
Proteomics Experimental Design
Sample Procurement
• plasma, serum• lysates Sample
Processing
• fractionate• deplete• digest…
Data Collection
• LC-MS/MS• MALDI, SELDI• 2D gel
Data Analysis
• univariant• multivariant• supervised or no?
Design decisions should be made based on sample limitations, experimental goals, and resources
Sample for Analysis• Optional Clean Up
Tag based method• Tag (ICAT)• Digest • Fractionate• Affinity capture• Optional sample
cleanup
Global digest
• Deplete • Fractionate• Digest• Optional sample
cleanup
Intact protein analysis• Deplete• Fractionate• Optional sample
cleanup
Analysis method
• LC-MS• MS/MS• MALDI
Analysis method• LC-MS• MS/MS• MALDI
Analysis method• 2D gel electrophoresis• FTMS, ETD/ CID• LC-MS/MS
(peptidomics)• MALDI/SELDI
Typical Proteomics Process Flow
� Complexity reduction = depletion, fractionation, tagging� MS analytical ‘aids’ = digestion
Serum or Plasma Sample Optional Clean Up (filtration)
Depletion of
IgG & HSA
Optional sample cleanup
Fractionate with
IEX (strong anion)
Analysis method
• 2D gel electrophoresis• FTMS, ETD/ CID• LC-MS/MS
(peptidomics)• MALDI/SELDI
Depletion Followed by Ion Exchange
Optional Denaturation / Protein interaction disruption
pH elution
Optional
digestion
� Ion Exchange (IEX)
� Affinity
� Mixed Mode Chromatography
� Size Exclusion (Gel Filtration)
� Pall Specialty Sorbents
Methods of Chromatography
Pall Chromatography Range
Ion Exchange
SizeExclusion
HIC
AndSpecialty
�DEAE, CM, SP Trisacryl® M/LS
�DEAE, SP Spherodex® LS
�QMA Spherosil® LS
�Q, S, DEAE, CM Ceramic HyperD® 20 and F
�Q and CM HyperZ
Affinity
�Heparin HyperD®
� Lysine HyperD®
�Protein A Ceramic Hyper D�Blue Trisacryl
®
M Affinity� IMACTM HyperCel
�Methyl HyperD®
�SDR HyperD®
�HA Ultrogel
�MEP Hypercel
�HEA/ PPA HyperCel
�Ultrogel AcA
�Trisacryl GF
� Ion Exchange Chromatography
� Q/ S/ DEAE/ CM Ceramic HyperD resins� Mustang Q/ S membranes
� Q (Quarternary Ammonium) = Strong Anion Exchanger
� S (Sulfonic Acid) = Strong Cation Exchanger� DEAE (Dextran) = Weak Anion Exchanger� CM (Carboxymethyl) = Weak Cation Exchanger
Ceramic HYPERD sorbents
In situ polymerization
Monomer intrusion
� Porous, non-compressible ceramic bead
� >0.2 µm (2000 Å) ‘pores’� In situ polymerization to form hydrogel
bead, containing the functional groups � CM, Q, S, DEAE IEX sorbents
“a gel in a shell”
Q/S/DEAE/CM Ceramic HyperD
� Features:� High Dynamic binding capacity at high flow rates� High-efficiency capture from dilute feedstock� Rigid, non compressible sorbent – easy to pack� Easy cleaning with NaOH� High speed, high capacity affinity preparative resins
for the purification of biological molecules by charge
Anion Exchange Dynamic Binding Capacity
• Anion Exchange: 5mg/ml BSA in 25 mM Tris pH 8.5, conductivity = 4-5 mS• ~0.85 ml column, run on Akta Explorer
Resinave bead
size
Slurry Volume % 10% 50% 10% 50%
Q HyperD F1 50 µ 125 129 114 122Q HyperD 20 20 µ 139 134 130 129Q (competitor A) 30 51 13 22Q (competitor B) 15 µ 75 76 66 40Q (competitor C) 42 62 15 33DEAE HyperD F1 50 µ 121 121 112 118DEAE (competitor A) 52 81 27 42DEAE (competitor C) 28 26 13 20
1 ml/min 4 ml/min
� Mustang ion exchange� 96-well format� Acrodisc format� Larger devices
NEW Membrane-based Chromatography at Pall
Fractionation of Human Serum in Mustang Q Anion Exchange Membrane in 96-Well Plate Format
� Compare flow through and eluate by 1D SDS-PAGE, reduced
� Consistent with expected IEX behavior, at pH 7, very few proteins captured, at pH 10 most proteins capture
� The presence of 150 mMNaCl in buffer reduces number of bound proteins
� Easier then using beads� Binding capacity relative to
membrane bed volume.
Effect of Loading pH and NaCl
pH 7.0 7.0 10.0 10.0 [NaCl] mM 150 0 150 0
250
150
100
75
50
37
25
15
20
10
kD
MW - FT E FT E FT E FT E FT E
Affinity Chromatography
� Separation using specific ligands� Reversible binding (buffer/ salt/ pH)
� Enchant Protein Depletion Kits� Blue Trisacryl M� Protein A Ceramic HyperD� IMAC Hypercel� Heparin HyperD� Lysine HyperD
• Albumin Depletion
• Albumin Fractionation
• Albumin & IgG Depletion
– Affinity Ligand based kit
Enchant™ Kits – Abundant Protein
Depletion
Abundant Proteins
� There are six abundant proteins that researchers often want to remove� Albumin
� IgG
� IgA� Transferrin� Anti-trypsin� Haptoglobin
Why Deplete High Abundant Proteins?
� Starting sample contains a mixture of both high abundance and low abundance proteins
� Proteins researchers are interested in are present in a complex sample
� Need to reduce the complexity of the sample� Proteins of interest are low molecular weight, low
abundant proteins
� First step in complexity reduction is the removal of high abundant proteins
� Without removing high abundant proteins, studying/identifying proteins of interest is like looking for a needle in a haystack
2DGE Plasma
Blue Trisacryl®M Affinity Sorbent
Structure of Blue Trisacryl M.
Typical applications:
• Albumin
• Vaccines• Interferons.• Purification of growth factors.• Isolation of DNA-dependent enzymes.• Purification of coagulation factors.• Purification of lipoproteins.
Ligand : CIBACRON BLUE (Blue TRISACRYL M)BASILEN BLUE ( Blue TRISACRYL PLUS LS)
Protein A Ceramic Hyper D
Protein A Sorbents : introduction
� Protein A is the industry standard for the capture step of antibodies, both in a laboratory and large-scale production.
� Generic, simple, very selective, allows direct recovery with minimal pre-treatment
� Several FDA approved Mabs processes for therapeutic usage refer to protein A sorbents.
� Typically, Protein A capture is followed by two « orthogonal » steps (ion exchange or HIC), to get rid of contaminants and remove traces of protein A.
Limitations of Protein A
� Leaching of Protein A� Cost� Variable binding capacity for different IgGs
� Species� Class� Sub-class� The need for Protein G
� Loss of protein activity over time due to regeneration in harsh conditions
Pall offer another alternative to Protein A and G
MEP HyperCel™
MEP chemistry
� Hydrophobic charge induction mechanism
Interaction of 4-MEP Ligand with Antibody
S
N
pKa = 4.8Hydrophobicinteraction
Adsorption at near-neutral pH
Desorption at pH 4
H
S
N
+++++++++
++++
++++
++++
Electrostatic Repulsion
pH% in (+)Form
4.8 50%
5.8 10%
Desorption at pH 4.0 – 5.8
IMAC HyperCel Resin
� For immobilised metal ion affinity chromatography
� Pre-fractionation method to increase resolution
� purify and collect tagged proteins ie His-tag, antibodies or phosphorylated proteins
� Can be charged with: Cu(II), Ni(II), Zn(II), Co(II), Ag(I), Ga(III), Zr(IIII) and Fe(III)
Ligand is tridentate IDA (Iminodiacetic acid) immobilisedon HyperCel base sorbent
� Size Exclusion Chromatography
� Also called gel filtration or gel permeation chromatography� Separates molecules according to size� Simple to use, non-denaturating method� Separates monomers from aggregates (i.e. IgG)� Desalting (buffer exchange) � Time consuming, low flow rates� LIMITATION: Final product is diluted
� Alternative is MWCO separation using ultrafiltration membrane
Ultrogel® AcA Product LineBioSepra Frac. range Excl. limit
sorbent (dalton) (dalton)
Ultrogel AcA202 1,000-15,000 22,000
Ultrogel AcA54 5,000-70,000 90,000
Ultrogel AcA44 10,000-130,000 200,000
Ultrogel AcA34 20,000-350,000 750,000
Ultrogel AcA22 100,000-1,200,000 3,000,000
Application of Pall Size Exclusion Sorbent
Sample Cleanup (Desalting)
� AcA 202 and GF05 can be used for desalting:
� very low non-specific interactions� ready to use, no gel swelling required� better resolution
� Desalting is one of the most commonly performed size exclusion steps
� Goal – remove small molecules (salt, free label)
� Using a spin device or filter plates avoids that common problem of sample dilution
� 5 mg/ml HSA in 1M NaCl onto 10 ml column, 1 ml fractions collected
� A280 measured the HSA coming through the column
� Conductivity was used to measure the removal of NaCl
Desalting in Gravity Flow Column
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0 5 10 15 20 25
Fraction number
Ab
so
rban
ce @
280 n
m
0
5
10
15
20
25
30
Co
nd
ucti
vit
y m
S/c
m
A280 nm NaCl
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 5 10 15 20 25
Fraction number
Ab
so
rba
nc
e @
28
0 n
m0
5
10
15
20
25
30
35
40
Co
nd
uc
tivit
y (
mS
/cm
)
AcA202 Resin GF05M
� 0.1 ml of 5 mg/ml HSA in 1M NaCl was loaded onto nanosep column (0.2ml resin)
� Minimal sample dilution is seen when a centrifugation protocol is used
� Measured conductivity indicates very efficient removal of NaCl
Desalting in Nanosep Spin Column
1.050.095Competitive
agarose
0.0210.10AcA 202
0.450.12GF-05M
Conductivity
(mS/cm)2
Volume recovered
(ml)1
Resin
Specialty sorbents
�SDR HyperD�Unique to Pall Life Sciences
Silica SurfaceSilica SurfaceSilica SurfaceSilica Surface
Hydrophobic PolymerHydrophobic PolymerHydrophobic PolymerHydrophobic Polymer
SDR HyperD ChemistrySDR HyperD ChemistrySDR HyperD ChemistrySDR HyperD Chemistry
SDR Mechanism of Detergent Binding
10 kDexclusion limit
Triton
TnBP
SDR HyperD ChemistrySDR HyperD ChemistrySDR HyperD ChemistrySDR HyperD Chemistry
SDR Mechanism of Detergent Binding
10 kDexclusion limit
ASB-14 Removal on SDR HyperD
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
0 10 20 30 40Fraction number
Ab
so
rban
ce @
595 n
m
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Ab
so
rban
ce @
280 n
m
A 595 nm, detergent A 280 nm, HSA
� 5 mg/ml HSA in 1% detergent onto 1 ml packed column
� After loading, 1% detergent in buffer was added until break through was seen
� Break through determined based on inhibition of dye binding assay at OD 595 nm
Dynamic Binding Capacity Determination
SDR HyperD Detergent Removal
Examples of Binding Capacity
28.0SDS + 0.1 M NaCl
15.0SDS
75.0CHAPS
70.0ASB-14 + 6M Urea / 2M Thiourea
60.0 mg/mlASB-14
SDR HyperDDetergent � 5 mg/ml HSA in 1% detergent onto 1 ml packed column
� After loading, 1% detergent in buffer was added until break through was seen
� Break through determined based on inhibition of dye binding assay at OD 595 nm
Removal of Detergents from Protein Solutions
Triton (DBC = 60-80 mg/ml)
Initial Conc. (ppm
Final Conc. (ppm)
Removal efficiency
10,000
<10
>99.9%
10,000
<10
>99.9%
10,000
340
95.2%
Detergent Protein solutions
AT-IIIIgG Bovine Serum
TnBP (DBC = 40-60 mg/ml)
Initial Conc. (ppm
Final Conc. (ppm)
Removal efficiency
5,000
<0.4
>99.99%
5,000
<0.4
>99.99%
5,000
3.8
99.92%
Enrichment & Prefractionation
Mustang Resin Recombinant
Protein
Blue tris acryl
IMAC
IEX AFFINITY
DesaltingDetergent/ Solvent Removal
GEL FILTRATIONMIXED MODE
Trisacryl
UltrogelSDR HyperCel
Protein &
Peptide
Enrichment
Mustang Q
Mustang S
Q Ceramic HD F
S Ceramic HD F
DEAE Ceramic HD F
CM Ceramic HD F
Protein A Ceramic HD F
Heparin HD M
Lysine HD
Blue Trisacryl M
IMAC Hypercel
SDR Hyper D
Trisacryl GF05M
Ultrogel AcA 202
Monoclonal Ab
Recombinant
proteins
IgG, Albumin
Purification / Depletion
Coagulation factors
Lipoproteins, GH
Glycoproteins
Tagged Biomolecule
Purification
Solvent &Detergent
RemovalDesalting &
Small molecule
removal
Sample prep tools for protein purification
AffinityIon
Exchange
Size Exclusion
Concentrate
Polishingaffinity
and/
or
and/ or
Summary of Resins
At this stage you are pulling out what you want and eliminating the junk.
Then, you may wish to fractionate what is left based on size – resolution.
Once you have isolated the right fraction, if using size exclusion you will need to concentrate the product using ultrafiltration or a polishing affinity resin step.
Useful Chromatography Formats for Biology
� 96 well plates� Medium through put small scale fractionation
for proteomics or purification� Small scale purification without expensive
chromatography systems or expertise� Purification development – scouting� Disposable – no chance for cross contamination
� Centrifugal devices (spin columns)� Small scale purification without expensive
chromatography systems or expertise� Disposable – no cross contamination
� Small to very large traditional column chromatography
High throughput sample processing in proteomic research requires the protein recovery be consistent from well to well in the filter plates.
As shown, the elution from 96 identical samples processed by minicolumns in a single AcroPrepfilter plate with 0.45 µm GHP membrane (PN 5030) was consistent from well to well as judged by the intensity of protein bands in SDS PAGE gels. In addition, the protein concentration of each eluted sample was quantified by BCA assay, giving a CV of 9.3%.
This result indicated superb well-to-well reliability of the AcroPrep 96 filter plate in processing multiple samples.
Protein Purification – Scouting Experiment
An example of superb well-to-well reliability using an
AcroPrep™ 96 filter plate with hydrophillic media as chromatography minicolumns
Uses of UltraFiltration Devices
� Desalting & size exclusion with MWCO� Omega UF membranes� Spin devices – Nanosep, Microsep, Macrosep,
Jumbosep� TFF – also for sample concentration- Minimate
� Limited to 2 fractions (retentate and filtrate)� Some proteins can be lost on/in the membrane
� Might be faster then bead based separations� Less likely to see sample dilution, possibly combine
with a concentration step
• Small scale purification without expensive
chromatography systems or expertise
– Varying Sizes • Nanosep®: less than 0.5 mL• Microsep™: 0.5 – 3.5 mL• Macrosep®: 3 –15 mL• Jumbosep™: 15 – 60 mL
– Ultrafiltration Devices• Desalting• Concentration• Buffer Exchange
– Microfiltration Devices
• Batch mode chromatography• Small, fast protein preps
Spin Filter Columns – Single Sample Format
Western Blotting
� What? When? Where?� Nitrocellulose
� BioTrace NT – pure unsupported Nitrocellulose � 0.45um PVDF
� BioTrace PVDF – strong hydrophobic interaction� 0.2um PVDF – increased sensitivity, lower burn-
through� FluoroTrans – N-terminal sequencing� FluoroTrans W – optimised for Western blots
� 96-well plate Acrowell – ELISPOT assays� BioTrace PVDF� BioTrace NT
Western Blotting - Troubleshooting
� Before you blame the membrane…� Has your protein transferred properly?� Have you checked your blocking solution?� Have you checked your primary antibody?� Have you checked your conjugate?� Do your detection reagents give ideal results?� Is your water actually ultrapure 18.2MOhm?� All this should be run with known working
standards.
• Biosepra® Chromatography Resins
• Enchant™ Protein Purification/Depletion/Fractionation Kits
• Nanosep®, Microsep™, Macrosep®, &Jumbosep™ Centrifugal Spin Filters
• AcroWell™ & AcroPrep™ Multi-well Filter Plates
• Minimate™ TFF Systems
• Acrodisc® Syringe Filter Columns
• BioTrace™ & FluoroTrans®
Blotting Membranes
Making Protein Sample
Preparation and Analysis Easy
LC Sample and Mobile Phase Filtration
� Traditional options� Hydrophilic Nylon/ PVDF – Aqueous/ some organics� Hydrophobic PTFE – Solvent/ Aggressive organics
� 0.45um standard� 0.2um recommended for smaller bead sizes� Introducing GHP
� Universal HPLC Membrane� Hydrophilic Polypropylene� Aqueous and Aggressive solvents� Low Protein binding
HPLC: Why should I filter?
� What Pore size?� 0.2um – Beads 3um and smaller� 0.45um – larger than 3um
� Mobile Phase/ Sample: Removing particulate/ bacteria from your column will extend the life of your column, saving you money
� Features to consider:� Accuracy of pore size� Extractables� Biomolecule binding
Thank You for
Your Attention!