1

Gel Filtration (GF) or

Size Exclusion Chromatography

(SEC)

2

Gel Filtration chromatography (GF)

Principles of GF

Fractionation range

Parameters for resolution optimization

Use of GF

Column performance

Troubleshooting

Examples

3

What is gel filtration?

Gel filtration is a technique of liquid chromatography which separates molecules according to their ratio (sizes, oligomeric state) Beads with pores of well-defined sizes: fractionation range Mobile phase: almost all kind of buffers

4

Gel structure Agarose

Dextran

A hypothetical structure for Superdex

Beads of defined porosity: fractionation range

The degree of cross-linking determines the size of the pores and therefore the

fractionation range of the resin

Unlike many other chromatographic procedures, size exclusion is not an adsorption

technique.

Void volume Vo

Volume of the gel matrix Vs

Pore volume Vi

5

Terms and explanations

Vo= Void volume: volume of the solution outside the beads, or

elution from very large molecules

Ve = the volume from the time the protein is placed until it

appears in the effluent

Vi = volume of the solution inside the beads = Vc - Vs - Vo

Vc = Total (geometric) volume of the column

Vt = Elution volume for very small molecules

2 3

Void volume Vo

Volume of the

gel matrix Vs

Pore volume Vi

1

Vo

Ve

Vt

Vc

https://www.youtube.com/watch?v=oV5VB5kO3tQ https://www.youtube.com/watch?v=E3z1wIImvHI https://www.youtube.com/watch?v=rPRbqYWlSEo

6

Steric exclusion

Molecules are excluded from the gel bead to different extents according to their sizes.

Gel bead

Largest molecules - excluded from pores, travel with the mobile phase, elute rapidly from column • The volume at which large molecules elute is called the void volume, Vo (same as the volume of solution that surrounds the beads)

Smallest molecules – enter the pores of the beads, are included in the matrix and retarded in their movement, spend most of the time in the stationary phase, elute last • The volume at which small molecules elute corresponds to Vt (total volume of solution surrounding (Vo) and inside the beads, Vs) Vt = Vo + Vs

Intermediate size molecules – spend different amounts of time both inside and outside the beads (partition between the mobile and stationary phase) • The volume at which intermed.molecules elute is called the elution volume (Ve) and depends on the partition of the molecule between the Vo and Vs which is proportional to the distribution coefficient (K) Ve = Vo + KVs

7

Constructing a selectivity curve

Kav 1

0 log (Mr)

Run standards and determine the elution volume for each

Calculate Kav values

Plot log (Mr) for each standard against the calculated Kav

Selectivity curve is usually moderately straight over the range Kav=0.1 to Kav=0.7

Extrapolate MW of your protein according to his Ve

ot

o

VV

VVeavK

8

Shape effects

Different fractionation

ranges for:

•Native, globular proteins

•Partially folded molecules

•Proteins inside detergent

micelle: MW of protein +

MW of micelle

Denatured proteins

Kav 1

0.7

0.1

log (Mr)

Native proteins

9

How to choose GF type

Kav 1

log (Mr)

Molecules with different shapes have different

selectivity curves

Linear polysaccharides

Globular proteins

RESIN FR Glob Prot FR Dextrans

Sephacryl S100 1-100 kDa ND

Sephacryl S200 5-250 kDa 1-80 kDa

Sephacryl S300 10-1500 kDa 2-400 kDa

Sephacryl S400 20-8000 kDa 10-2000 kDa

Protein 1: 30kDa Protein 2: 80kDa

Vo Vt

10

Gel Filtration chromatography (GF)

Principles of GF

Fractionation range

Parameters for resolution optimization

Use of GF

Column performance

Troubleshooting

Examples

11

Incr

easi

ng

excl

usi

on

lim

it

Results depend on selectivity (fractionation range)

Sephacryl S-100

40 80 120 ml

Sephacryl S-300

Sephacryl S-200 BSA Cyt C

IgG b-L Cytidine

AU280

Better for larger proteins

Better for smaller proteins

Best for these proteins

RESIN FR Glob Prot FR Dextrans

Sephacryl S100 1-100 kDa ND

Sephacryl S200 5-250 kDa 1-80 kDa

Sephacryl S300 10-1500 kDa 2-400 kDa

Sephacryl S400 20-8000 kDa 10-2000 kDa

12

High efficiency can compensate for low selectivity.

If selectivity is high, low efficiency can be tolerated (if large peak volume is acceptable).

Resolution depends on efficiency and selectivity

Low selectivity

High selectivity

high efficiency

low efficiency

high efficiency

low efficiency

13

14

Efficiency

Efficiency depends on:

Particle size of matrix

Particle size distribution of matrix

Packing quality of the column

Sample (volume, purity and viscosity)

Flow rate (more important for bigger beads)

15

Peak width depends on particle size

Superdex Peptide 13-15 µm Superdex 30 prep grade 24-44 µm

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 10 20 30 40 50

Retention time (min)

AU214

0

0.01

0.02

0.03

0.04

0.05

0.06

0 10 20 30 40 50

Retention time (min)

AU214

16

1 x Superdex® Peptide HR 10/3000 2 x Superdex® Peptide HR 10/30

Resolution depends on column length Increasing column length increases resolution

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0 5 10 15 20 25

Retention time (min)

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0 10 20 30 40 50

Retention time (min)

AU214

0

50

100

150

mAU

40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 ml

0

50

100

150

mAU

60.0 70.0 80.0 90.0 100.0 110.0 ml

Superdex® Peptide 60 x 1.6cm ~ 120ml Superdex® Peptide 100 x 1.6cm ~ 200ml

SUMO-Atox1

SUMO-Atox1

SUMO

SUMO

Atox1

Atox1

Michal Shoshan from Edith Tshuva lab.

17

Column: Superdex® Peptide HR 10/30

Resolution depends on sample volume

25µl

0

0.02

0.04

0.06

0.08

0.1

0 5 10 15 20 25

Retention volume (ml)

AU214

200 µl

200 µl

400 µl

0

0.02

0.04

0.06

0.08

0 5 10 15 20 25

Retention volume (ml)

AU214

400 µl

0

0.02

0.04

0.06

0.08

0.1

0 5 10 15 20 25

Retention volume (ml)

A214

25 µl

18

Why use gel filtration?

Group separations: Desalting, Buffer exchange, Removing reagents (replace dialysis)

Purification of proteins and peptides: complex samples, monomer/dimer

QC: Estimation size & size homogeneity

Protein-Protein Interaction

19

Desalting proteins

Desalting in a simple column

Column:

Sample:

Buf fer:

PD-10

HSA, 25 mg

NaCl 0.5M

HSA NaCl

volume

Volume for desalting: up to 25% column volume

20

Use in group separations

Adjusting pH, buffer type, salt

concentration during sample

preparation, e.g. before an assay.

Removing interfering small

molecules: EDTA, Gu.HCl, etc

Removing small reagent molecules,

e.g. fluorescent labels, radioactive

markers.

Gravity Desalting Columns

Multi Spin Desalting Columns

FPLC Desalting Columns

Spin Desalting Columns

21

HiTrapDesalt10ml001:1_UV1_280nm HiTrapDesalt10ml001:1_Cond HiTrapDesalt10ml001:1_Fractions HiTrapDesalt10ml001:1_Inject HiTrapDesalt10ml001:1_Logbook

0

100

200

300

400

500

600

700

800

mAU

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

HiTrapDesalt10ml002:1_UV1_280nm HiTrapDesalt10ml002:1_Cond HiTrapDesalt10ml002:1_Fractions HiTrapDesalt10ml002:1_Inject HiTrapDesalt10ml002:1_Logbook

0

200

400

600

800

1000

mAU

0.0 5.0 10.0 15.0 ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Waste 17 18 Waste

Desalting in the presence of buffer + 250mM NaCl

Desalting in the presence of buffer + 100mM NaCl

OD 280nm

Conductivity

Use buffer that avoid protein precipitation

Gali Prag

22

Fractionation of multiple components

Separate multiple components in a sample on the basis of differences on their size

Best results with samples that contains few components or partially purified samples

(polishing step) : Not recommended for proteins close by MW

Limited sample volume (0.5-4% of total column volume). Not so suitable if the

sample volume is large

Flow-rate limitation : Time consuming

Removes higher oligomeric states and other aggregates

Protein elutes with the column equilibration buffer

23

Separating dimer and oligomers from monomer

Column: Superdex 75 HR 10/30 Sample: A special preparation of rhGH in distilled water

0.025

0.05

Oligomer

Monomer

ime (min)

Dimer

T 10 20 V O V C

280 nm

A

11/26/2015 24

Case study: HLT-p53CT- Affinity start with pellet of 1.5L culture

Ni-Sepharose FF 14ml

HLTp53CTNiNTA16ml004:1_UV1_280nm HLTp53CTNiNTA16ml004:1_UV2_260nm HLTp53CTNiNTA16ml004:1_Conc HLTp53CTNiNTA16ml004:1_Fractions HLTp53CTNiNTA16ml004:1_Inject HLTp53CTNiNTA16ml004:1_Logbook

0

500

1000

1500

2000

2500

mAU

200 250 300 350 400 450 ml

F4 Waste 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Load + 10cv 0%B + 3cv 8%B + 4cv 15%B + 4cv 100%B

POOL 17-22: 3.5OD x 35ml ~ 276mg

11/26/2015 25

HLT-p53CT- Cation Exchange after TEV

protease cleavage ON 4ºC SP-Sepharose FF 5ml HLTp53CTHiTrapSP5mlml005:1_UV1_280nm HLTp53CTHiTrapSP5mlml005:1_UV2_260nm HLTp53CTHiTrapSP5mlml005:1_Cond HLTp53CTHiTrapSP5mlml005:1_Conc

HLTp53CTHiTrapSP5mlml005:1_Fractions HLTp53CTHiTrapSP5mlml005:1_Inject HLTp53CTHiTrapSP5mlml005:1_Logbook

0

50

100

150

mAU

500 550 600 650 700 ml

F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

26

p53C-Terminal - Polishing column Column: Sephacryl S100 prep. 960 x 26mm (~500ml) 6ml/fract.

Ni column – TEV protease cleavage ON – dilution – CEIX – concentration & GF

HLTp53CTSephacrylS100of500ml004:1_UV1_280nm HLTp53CTSephacrylS100of500ml004:1_UV2_260nm HLTp53CTSephacrylS100of500ml004:1_Fractions HLTp53CTSephacrylS100of500ml004:1_Inject HLTp53CTSephacrylS100of500ml004:1_Logbook

0

50

100

150

mAU

150 200 250 300 350 400 ml

F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Fractions around 23 and 32 are higher MW impurities

POOL 7-14

Ronen Gabizon from Assaf Friedler lab.

27

Quality control: Use for size estimation / oligomeric state

Gives an estimate of molecular size in native or denative solution (Guanidine HCl, urea, detergents)

MW of globular protein using native buffers (Precision is not so good)

Oligomeric state of the protein / homogeneity / complex

Complementary information to PAGE-SDS

Size exclusion chromatograph in line with multi angle light scattering ,

added value to characterize proteins mass and shape in native solution

conditions

Calculating Mw and radius from the light scattering equations – much

more accurate.

Calculate the Mw during the elution peaks- detect homogeneity sample.

Detect low amount of aggregation – large molecules amplify the

intensity of LS.

Useful for protein/protein or protein/ligand interaction

SEC-MALS: Size Exclusion Chromatography - Multi Angle Light Scattering

29

Refolding by Dilution with Non Detergent Sulfo Betaine

0.0

5.0

10.0

15.0

mAU

0.0 5.0 10.0 15.0 ml

Refolding with 1MNDSB at 0.1mg/ml

0.0

10.0

20.0

30.0

40.0

mAU

0.0 5.0 10.0 15.0 ml

refolding with 1M NDSB at 0.2mg/ml

0.0

5.0

10.0

15.0

20.0

mAU

0.0 5.0 10.0 15.0 ml

refolding with 1M NDSB at 0.5mg/ml

0.0

10.0

20.0

30.0

mAU

0.0 5.0 10.0 15.0 ml

Refolding with 1M NDSB at 1mg/ml

Multimer Monomer

30

Complex formation: Leptin and Leptin Receptor

Superdex75prep002:1_UV3_220nm Superdex75prep002:1_Fractions Superdex75prep002:1_Inject Superdex75prep002:1_UV3_220nm1 Superdex75prep002:1_UV3_220nm2 Superdex75prep002:1_Logbook

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

mAU

100 150 200 250 ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Waste

Superdex 75 160x1.6cm column - Buffer: 20mMTrisHCl pH8.0 50mMNaCl 0.02%NaN3

Receptor alone

Leptin alone

Complex: Leptin + Receptor

31

Gel Filtration chromatography (GF)

Principles of GF

Fractionation range

Parameters for resolution optimization

Use of GF

Column performance

Troubleshooting

Examples

32

Increasing resolution

Choose appropiate fractionation range

Increase column volume (Connect two columns)

Reduce the flow rate

Change to a gel with smaller beads (higher efficiency)

Reduce the sample volume / protein quantity

Check the column efficiency

Clean and/or re-pack

SUMO alone and ATOX1

ATOX1 alone

Optimization of ATOX1 purification

Michal Shoshan from Edit Tshuva lab

SUMO-Atox1 IMAC purification

ATOX1: SUMO protease treatment after IMAC purification. Concentration by UF instead of AS precipitation. Load on 200ml

Superdex 30 column (20mM MES pH 6.0 + 150mM NaCl)

ATOX1: SUMO protease treatment after IMAC purification. Ammonium Sulphate precipitation. Load on 120ml Superdex 30

column (20mM MES pH 6.0 + 150mM NaCl)

ATOX1: SUMO protease treatment after IMAC purification. Concentration by UF in the presence of 4M NaCl. Load on 200ml

Superdex 30 column (20mM MES pH 6.0 + 250mM NaCl)

SUMO alone

ATOX1 alone

0

50

100

150

mAU

60.0 70.0 80.0 90.0 100.0 110.0 ml

chelating2mlAir Imp7Beta001 18 12 12001:1_UV1_280nm chelating2mlAir Imp7Beta001 18 12 12001:1_UV2_260nm chelating2mlAir Imp7Beta001 18 12 12001:1_Conc chelating2mlAir Imp7Beta001 18 12 12001:1_Fractions chelating2mlAir Imp7Beta001 18 12 12001:1_Logbook

0

20

40

60

80

100

%B

120.0 130.0 140.0 150.0 160.0 170.0 180.0 190.0 ml

F4 1 F2 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3132

Fractions 16-22 Peak-833 mAU

100%B

20%B

15%B

Chelating2mlAir, Imp7598 ImpBeta1-442 from 1L 17C ON 19.12.12

Imp7BetaSuperose12prepar200ml 18 12 12001:1_UV1_280nm Imp7BetaSuperose12prepar200ml 18 12 12001:1_UV2_260nm Imp7BetaSuperose12prepar200ml 18 12 12001:1_Fractions Imp7BetaSuperose12prepar200ml 18 12 12001:1_Inject Imp7BetaSuperose12prepar200ml 18 12 12001:1_Logbook

0

20

40

60

80

100

120

140

mAU

60 80 100 120 140 160 ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Extra ImpBeta Peak - 106ml

Complex Peak - 87ml

Aggregate Peak - 65ml

Superose12prepar200ml Imp7 598 ImpBeta1-442 + Tween20 0.01% after Ni 20/12/12 Imp7BetaSuperdex75prep320ml 25 04 12001:1_UV1_280nm Imp7BetaSuperdex75prep320ml 25 04 12001:1_UV2_260nm Imp7BetaSuperdex75prep320ml 25 04 12001:1_Fractions Imp7BetaSuperdex75prep320ml 25 04 12001:1_Logbook

-10.0

0.0

10.0

20.0

30.0

40.0

50.0

60.0

mAU

80 100 120 140 160 180 200 ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Peak2: 182 ml

Peak1: 137 mlPeak agregate: 115 ml

Imp7 (598-C) + Imp Beta (1-442) after Ni and conc - Superdex75prep320ml 25.04.12

Optimization of Complex Formation

Strategy: Co-purification of two proteins (separately

expressed) Complex stabilization: 0.01% Tween-20

Capture: IMAC column Nadav Komornik / Oded Livnah lab

Superose 12 200ml column Superose 12 320ml column 0.01% Tween-20 through purification

Aggregate

Complex Free protein

Increasing resolution - Example: Pegylated protein 120617M1605Superose12Anal001:1_UV1_280nm 120617M1605Superose12Anal001:1_UV2_260nm 120617M1605Superose12Anal001:1_Fractions 120617M1605Superose12Anal001:1_Inject 120617M1605Superose12Anal001:1_Logbook

0

50

100

150

mAU

5.0 10.0 15.0 20.0 ml

F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Waste

120618Superose12Prep3columns500ml001:1_UV1_280nm 120618Superose12Prep3columns500ml001:1_UV2_260nm 120618Superose12Prep3columns500ml001:1_Fractions 120618Superose12Prep3columns500ml001:1_Inject 120618Superose12Prep3columns500ml001:1_Logbook

0

50

100

150

mAU

100 150 200 250 300 350 ml

2 3 4 5 6 7 8 9 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93Superose 12 analytical 30 x 1cm = 23ml column

Load : ~1mg protein Superose 12 preparative

3 tandem columns 250 x 1.6cm = 502ml column Load : ~25mg protein / 5ml

Vo

1 2 3

How can we get better separation between 2 and 3 ??

Can we scale-up protein loading to separate 1 from 2 and 3 ??

36

Column size

Desalting and other group separations

Volume four times the expected sample volume

Length is not so important

Fractionation

Volume ~0.5-4% times the expected sample volume

Sample volume can be increase if resolution is OK

Length 30-100 cm or more (depends of the resolution)

37

Why choose gel filtration?

Advantage

Separates by size. Complementary to IEX and HIC

Very gentle, high yields

Works in any buffer solution

Removes aggregates

Fast for buffer exchange

Mostly use in a final polishing step

Mandatory for QC

Complementary results than PAGE-SDS

Disadvantage

Limited sample volume

Poor resolution in a complex mixture

Flow-rate limitation – time consuming

Elution of diluted sample

Poor selectivity compared with SDS-PAGE

Not efficient in capture or intermediate steps

38

Troubleshooting

Lower yield than expected

Protease degradation of the protein

Adsorption to filter, valves or top of the column

Non-specific adsorption

Sample precipitate

MW of protein is not as expected

Oligomerization state of the protein is different

Protein bounds to another protein or complex

Unfolded or naturally unfolded protein

Protein has changed during storage

Ionic or Hydrophobic interactions between protein and matrix

Protein precipitate

Very broad peak elution

Different oligomeric states or protein aggregation

Sticky protein

Non specific adsorption to matrix

Protein is part of complex with different sizes

Overloading

Peak of interest is poorly resolved

Sample volume is too high

Short column

Poor selectivity or efficiency of the column

Flow rate too high

Column is dirty or not well packed

Viscous sample

39

Some molecules are highly hydrophobic, making them incompatible with fractionation via size-

exclusion chromatography (SEC). Field flow fractionation (FFF) separates macromolecules and

nanoparticles by size without a stationary phase, eliminating most of the non-ideal surface

interactions prevalent in SEC.

In an Asymmetric-Flow FFF separation channel,

macromolecules and nanoparticles are gently

pushed against a semipermeable membrane by

crossflow. Smaller particles diffuse back up towards

the center of the channel. Laminar channel flow

induces a parabolic flow velocity profile, causing

smaller particles to elute earlier.

Field flow fractionation (FFF)

40

Inhibition of HIV-1 Replication by Modulating the Oligomerization Equilibrium of the Viral Integrase

Zvi Hayouka. et al. PNAS 104 (20): 8316-8321 (2007) Friedler lab

The effect of ligand binding on the oligomeric state of Integrase

IN

IN + LEDGF/p75 361-370

IN + LEDGF/p75 361-370 (2h incubation)

IN + DNA LTR

IN + DNA LTR +

LEDGF/p75 361-370

Analytical gel filtration(superose 12)

200

125

9565Molecular weight

(gr/mol)

41

HTL435aaSuperdex200prep500mlB005:11_UV3_220nm HTL435aaSuperdex200prep500mlB005:11_Logbook

0

50

100

150

200

mAU

100 150 200 250 300 350 400 ml

NATIVELY UNFOLDED PROTEIN Case study: HTL - A natively unfolded proline-rich domain in ASPP2 that regulates its protein interactions by intramolecular binding to the Ank-SH3 domains.

Shahar Rotem et al. JBC Friedler lab

660 440 232 156 67 kDa

HTL 435aa

FoldIndex©: a simple tool to predict whether a given protein sequence

is intrinsically unfolded. Jaime Prilusky, Clifford E. Felder, Tzviya

Zeev-Ben-Mordehai, Edwin Rydberg, Orna Man, Jacques S.

Beckmann, Israel Silman, and Joel L. Sussman, 2005, Bioinformatics.

435aa

MW: 49.7kD

Superdex 200 prep. 100x2.6cm :

trimer ??

Analytical ultracentrifugation:

monomer