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Gel filtration

Principles and Methods

Handbooks

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Gel filtration Principles and Methods

Contents Introduction (7)

Chapter.1.

Gel.filtration.in.practice (9)

Introduction (9)

Purification by gel filtration (9)

Group separation (10)

High-resolution fractionation (11)

Rapid purity check and screening (12)

Resolution in gel filtration (12)

Sample volume and column dimensions (12)

Media selection (14)

Sample and buffer preparation (17)

Sample buffer composition (17)

Sample concentration and viscosity (17)

Sample volume (19)

Buffer composition (19)

Denaturing (chaotropic) agents and detergents (19)

Column and media preparation (20)

Sample application (21)

Elution and flow rates (21)

Controlling flow (23)

Method development for high resolution fractionation (23)

Maintenance of gel filtration columns (24)

Equipment selection (24)

Scaling up (24)

BioProcess Media for large-scale production (25)

Troubleshooting (26)

Chapter.2.

Superdex:.the.first.choice.for.high.resolution,.short.run.times,.and.high.recovery (31)

Separation options (33)

Separation examples (34)

Performing a separation (39)

First time use or after long-term storage (39)

Cleaning (40)

Removing severe contamination (40)

Media characteristics (41)

Chemical stability (41)

Storage (41)

Chapter.3.

Superose:5e0e3db65022aaea988f0f2dboratory.scale (43)

Separation options (45)

Separation examples (45)

Performing a separation (46)

First time use or after long-term storage (46)

Cleaning (47)

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Media characteristics (47)

Chemical stability (48)

Storage (48)

Chapter.4.

Sephacryl:.fast,5e0e3db65022aaea988f0f2dboratory.and.industrial.scale (49)

Separation options (52)

Separation examples (53)

Performing a separation (54)

First time use or after long-term storage (54)

Cleaning (55)

To remove severe contamination (55)

Media characteristics (55)

Chemical stability (56)

Storage (56)

Chapter.5.

Sephadex:.desalting,.buffer.exchange.and.sample.clean.up (57)

Separation options (58)

Separation examples (62)

Performing a separation (63)

General considerations (63)

Small-scale desalting of samples (63)

Desalting larger sample volumes using HiTrap and HiPrep columns (63)

Buffer preparation (64)

Sample preparation (64)

Buffer exchange (64)

HiTrap Desalting columns (64)

Manual purification with a syringe (65)

Simple desalting with ?KTAprime plus (66)

Desalting on a gravity-feed PD-10 column (67)

Buffer Preparation (67)

Optimization of desalting (67)

Scale-up and processing larger sample volumes (68)

Increasing sample loading capacity from 1.5 ml up to 7.5 ml (69)

Increasing sample loading capacity from 15 ml up to 60 ml (69)

For sample volumes greater than 60 ml (70)

Media characteristics (70)

Column Packing (71)

Cleaning (71)

Chemical stability (71)

Storage (71)

Chapter.6.

Sephadex.LH-20.–5e0e3db65022aaea988f0f2danic.solvents (73)

Media characteristics (73)

Separation examples (73)

Packing a column (75)

Performing a separation (76)

Cleaning (76)

Chemical stability (77)

Storage (77)

Transferring Sephadex LH-20 from aqueous solution to organic solvents (77)

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Chapter.7

Gel.filtration.in.theory (79)

Defining the process (79)

Selectivity curves and media selection (81)

Resolution (82)

Chapter.8.

Gel.filtration.in.a.purification.strategy (85)

The purification strategy according to CIPP (85)

Gel filtration as a polishing step (86)

monoclonal antibody (87)

Purification of humanized IgG

4

Appendix.1.

Column.packing.and.preparation (89)

Columns for packing gel filtration media (89)

Checking column efficiency (90)

Column packing for high resolution fractionation using

Superdex prep grade and Sephacryl High Resolution (91)

Column packing for group separations using Sephadex (93)

Controlling flow (95)

Appendix.2.

Sephadex.and.Darcy’5e0e3db65022aaea988f0f2dw (96)

Appendix.3.

Sample.preparation (97)

Sample clarification (97)

Centrifugation (97)

Filtration (97)

Desalting (97)

Denaturation (98)

Precipitation and resolubilization (98)

Ammonium sulfate precipitation (99)

Removal of lipoproteins (101)

Appendix.4.

Selection.of.purification.equipment (102)

Appendix.5.

Converting.from.linear.flow.(cm/h).to.volumetric.flow.rates.(ml/min).and.vice versa (103)

From linear flow (cm/h) to volumetric flow rate (ml/min) (103)

From volumetric flow rate (ml/min) to linear flow (cm/hour) (103)

From ml/min to using a syringe (104)

Appendix.6.

Conversion.data (105)

Proteins (105)

Nucleic Acids (105)

Column pressures (105)

Appendix.7.

Amino.acids.table (106)

Appendix.8..

Analysis.and.characterization (108)

Protein detection and quantification (108)

Purity check and protein characterization (108)

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Purity (108)

Characterization (109)

Appendix.9.

Storage.of.biological.samples (110)

General recommendations (110)

Common storage conditions for purified proteins (110)

Appendix.10.

Molecular.weight.estimation.and.molecular.weight.distribution.analysis (111)

Performing a molecular weight determination (113)

Product.index (115)

Related.literature (116)

5e0e3db65022aaea988f0f2drmation (117)

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18-1022-18 AK 7Introduction

Biomolecules are purified using chromatography techniques that separate them according to differences in their specific properties, as shown in Figure 1.

Property

Technique Size

Gel filtration (GF), also called size exclusion chromatography (SEC)Hydrophobicity

Hydrophobic interaction chromatography (HIC) Reversed phase chromatography (RPC)Charge

Ion exchange chromatography (IEX)Biorecognition (ligand specificity)

Affinity chromatography (AC)Isoelectric point

Chromatofocusing (CF)

Fig.1..Schematic drawing of separation principles in chromatography purification. From left to right: GF, HIC, IEX, AC, and RPC.Since the introduction of Sephadex? more than 50 years ago, gel filtration has played a key role in the purification of proteins and enzymes, polysaccharides, nucleic acids and other biological macromolecules. Gel filtration is the simplest and mildest of all the chromatography techniques and separates molecules on the basis of differences in size. The technique can be applied in two distinct ways:

1. Group separations: the components of a sample are separated into two major groups

according to size range. A group separation can be used to remove high or low molecular weight contaminants (such as phenol red from culture fluids) or for desalting and buffer exchange.

2. High resolution fractionation of biomolecules: the components of a sample are separated

according to differences in their molecular size. High resolution fractionation can be used to isolate one or more components, to separate monomers from aggregates, or to perform a molecular weight distribution analysis.

Gel filtration can also facilitate the refolding of denatured proteins by careful control of changing buffer conditions.

This handbook describes the use of gel filtration for the purification and separation of biomolecules, with a focus on practical information for obtaining the best results. The media available, selection criteria and examples with detailed instructions for the most common applications are included, as well as the theoretical principles behind the technique. The first step towards a successful separation is to select the correct medium and this handbook focuses on the most up-to-date gel filtration media and prepacked columns. Symbols

this symbol indicates general advice to improve procedures or recommend

action under specific situations.

this symbol denotes mandatory advice and gives a warning when special

care should be taken.

highlights chemicals, buffers and equipment.

outline of experimental protocol.

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Chapter 1

Gel filtration in practice

Introduction

Gel filtration (also referred to as size exclusion chromatography, SEC) separates molecules according to differences in size as they pass through a gel filtration medium packed in

a column. Unlike ion exchange or affinity chromatography, molecules do not bind to the chromatography medium so buffer composition does not directly affect resolution (the degree of separation between peaks). Consequently, a significant advantage of gel filtration is that conditions can be varied to suit the type of sample or the requirements for further purification, analysis or storage without altering the separation.

Gel filtration is well suited for biomolecules that may be sensitive to changes in pH, concentration of metal ions or co-factors and harsh environmental conditions. Separations can be performed in the presence of essential ions or cofactors, detergents, urea, guanidine hydrochloride, at high or low ionic strength, at 37°C or in the cold room according to the requirements of the experiment. Purified proteins can be collected in any chosen buffer.

This chapter provides general guidelines applicable to any gel filtration separation. A key step towards successful separation is selecting the correct medium; this handbook includes guides to the most up-to-date gel filtration media and prepacked columns. Application examples and product-specific information are found in Chapters 2 to 6.

Purification by gel filtration

To perform a separation, gel filtration medium is packed into a column to form a packed bed. The medium is a porous matrix of spherical particles with chemical and physical stability and inertness (lack of reactivity and adsorptive properties). The packed bed is equilibrated with buffer which fills the pores of the matrix and the space between the particles. The liquid inside the pores, or stationary phase, is in equilibrium with the liquid outside the particles, or mobile phase. Samples are eluted isocratically so there is no need to use different buffers during the separation. However, a wash step using the running buffer is usually included at the end of a separation to remove molecules that may have been retained on the column and to prepare the column for a new run.

Gel filtration can be used directly after ion exchange, chromatofocusing, hydrophobic interaction, or affinity, since the buffer composition will not generally affect the final separation. For further details on using gel filtration in a purification strategy, refer to Chapter 8.

Figure 1.1 illustrates the separation process of gel filtration and the theory for this process is described in Chapter 7.

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Fig.1.1..Process of gel filtration (A) Schematic picture of a bead with an electron microscopic enlargement. (B) Schematic drawing of sample molecules diffusing into bead pores. (C) Graphical description of separation I. Sample is applied on the column, II. The smallest molecule (yellow) is more delayed than the largest molecule (red). III. The largest molecule is eluted first from the column. Band broadening causes significant dilution of the protein zones during chromatography.

(D) Schematic chromatogram.

Group.separation

Gel filtration is used in group separation mode to remove small molecules from a group of larger molecules and as a fast, simple solution for buffer exchange. Small molecules such

as excess salt or free labels are easily separated from larger molecules. Samples can be prepared for storage or for other chromatography techniques and assays. Gel filtration in group separation mode is often used in protein purification schemes for desalting and buffer exchange. Sephadex G-10, G-25 and G-50 are used for group separations. Large sample volumes, up to 30% of the total column volume (packed bed), can be applied at high flow rates using broad, short columns. Figure 1.2 shows the chromatogram (elution profile) of a typical

group separation. Large molecules are eluted in or just after the void volume, V

o , as they pass

through the column at the same speed as the flow of buffer. For a well-packed column the void

volume is equivalent to approximately 30% of the total column volume. Small molecules such

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18-1022-18 AK 11as salts that have full access to the pores move down the column, but do not separate from each other. These molecules usually elute just before one total column volume, V t , of buffer has passed through the column. In this case the proteins are detected by monitoring their UV absorbance, usually at 280 nm, and the salts are detected by monitoring the conductivity of the buffer.

Column:

HiTrap? Desalting 5 ml Sample: (Histidine)6 protein eluted from HiTrap Chelating HP with sodium phosphate 20 mM, sodium chloride 0.5 M,

imidazole 0.5 M, pH 7.4

Buffer:

Sodium phosphate 20 mM, sodium chloride 0.15 M, pH 7.0void volume V o , total column volume V t

0.05

0.10

0.15

A 280 nm

Fig.1.2..Typical chromatogram of a group separation. UV (protein) and conductivity (salt) detection enable pooling of the desalted fractions and facilitate optimization of the separation.

Refer to Chapter 5, p 57 for detailed information on how Sephadex is used in group separation of high and low molecular weight substances in applications like desalting, buffer exchange, and sample clean up.

Refer to Chapter 7 for detailed information on the theory of gel filtration.

High-resolution.fractionation

Gel filtration is used in fractionation mode to separate multiple components in a sample on the basis of differences in their size. The goal may be to isolate one or more of the components, or to analyze the molecular weight distribution in the sample. The best results for high resolution fractionation will be achieved with samples that originally contain few components or with samples that have been partially purified by other chromatography techniques to eliminate most of the unwanted proteins of similar size.

High-resolution fractionation by gel filtration is well suited for the final polishing step in a purification scheme. Monomers are easily separated from aggregates. Samples can be transferred to a suitable buffer for assay or storage.

Rapid.purity.check.and.screening

Superdex? is a high resolution gel filtration medium. Superdex 75 5/150 GL and Superdex

200 5/150 Gl are short columns with small bed volumes and are suitable for rapid protein homogeneity analyses or purity checks. They save time when screening many samples, and require less buffer and sample than longer columns. However, when using the same media, shorter columns give lower resolution than longer columns.

Resolution.in.gel.filtration

The success of gel filtration depends primarily on choosing conditions that give sufficient selectivity and counteract peak broadening effects during the separation. After selection

of gel filtration medium, sample volume and column dimensions are the two most critical parameters that will affect the resolution of the separation.

The final resolution is influenced by many factors, see Table 1.1. The molecular weight range over which a gel filtration medium can separate molecules is referred to as the selectivity of the medium (see fractionation range guide for gel filtration media on page 16). Resolution is a function of the selectivity of the medium and the efficiency of that medium to produce narrow peaks (minimal peak broadening), as illustrated in Chapter 7, Figure 7.7.

Table.1.1..Factors that influence resolution

Medium-related factors Particle size

Particle uniformity

Match between pore size and analyte size

Column-related factors Bed height

Column packing quality

Experimental-related factors Flow rate

Sample volume

Viscosity

Sample.volume.and.column.dimensions

The sample volume can be expressed as a percentage of the total column volume (packed bed). Smaller sample volumes help to avoid overlap if closely spaced peaks are eluted. Figure 1.3 illustrates how sample volume can influence a high resolution fractionation.

For group separations, use sample volumes up to 30% of the total column volume.

For high resolution fractionation, a sample volume from 0.5% to 4% of the total column volume is recommended, depending on the type of medium used. For most applications the sample volume should not exceed 2% to achieve maximum resolution. Depending on the nature of the specific sample, it may be possible to load larger sample volumes, particularly if the peaks of interest are well resolved. This can only be determined by

experimentation.

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0.050.100.150.200.250.00

A nm nm

0.05

0.10

0.15

0.00

5.010.015.020.025.0A 2800.05

0.10

0.00

5.010.015.020.025.0A 280Fig.1.3..Influence of sample volume on resolution (Superdex 200 HR 10/30 is replaced with Superdex 200 10/300 GL).

The ratio of sample volume to column volume influences resolution, as shown in Figure 1.4, where higher ratios give lower resolution. Column volumes are normally selected according to the sample volumes to be processed. Since larger sample

volumes may require significantly larger column volumes, it may be beneficial to repeat the separation several times on a smaller column and pool the fractions of interest or concentrate the sample (see Appendix 3 on sample preparation).

For analytical separations and separations of complex samples, start with a sample volume of 0.5% of the total column volume. Sample volumes of less than 0.5% dot not normally improve resolution.

Concentrating samples can increase the capacity of a gel filtration separation. Avoid concentrations above 70 mg/ml protein as viscosity effects may interfere with the separation.

Sample dilution is inevitable since diffusion occurs as sample passes through the column. To minimize sample dilution, use a sample volume that gives the resolution required between the peaks of interest.

Column: Superdex 200 HR 10/30 (V t : 24 ml)Sample:.

M r .Conc..(mg/ml)

Thyroglobulin 669 000 3 Ferritin 440 000 0.7 IgG

150 000 3 Transferrin 81 000 3 Ovalbumin 43 000 3 Myoglobin 17 600 2 Vitamin B12 1 355 0.5 Total

15.2

Sample.load:.

A) 25 μl (0.1% × V t ) B) 250 μl (1% × V t )

C) 1000 μl (4.2% × V t )

Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0Flow.rate:

1.0 ml/min (76.4 cm/h)

A)

B)

C)

14 18-1022-18 AK Column: HiLoad? 16/60 Superdex 200 prep grade

Sample:

Solution of transferrin (M r 81 000) and IgG (M r 160 000) by equal weight

Sample.concentration: 8 mg/ml

Buffer: 50 mM NaPO 4, 0.1 M NaCl, pH 7.2

Flow.rate:

1 ml/min (30 cm/h)

Sample volume (% of column volume)

0.5

1.51.0

R e s o l u t i o n , R s Fig.1.4..Influence of ratio of sample volume to column volume on the resolution of transferrin and IgG on prepacked HiLoad 16/60 Superdex 200 prep grade. Resolution is defined in Chapter 7.

The height of the packed bed affects both resolution and the time taken for elution. The resolution in gel filtration increases with the square root of bed height. Doubling the bed height gives an increase in resolution equivalent to √2 = 1.4 (40%). For high resolution and

fractionation, long columns will give the best results. Sufficient bed height together with a low flow rate allows time for ‘intermediate’ molecules to diffuse in and out of the matrix and give sufficient resolution.

If a very long column is necessary, the effective bed height can be increased by using columns, containing the same media, coupled in series.

Refer to Chapter 7 for detailed information on the theory of gel filtration.

Media selection

Today’s gel filtration media cover a molecular weight range from 100 to 80 000 000, separating biomolecules from peptides to very large proteins and protein complexes.

The selectivity of a gel filtration medium depends solely on its pore size distribution and is

described by a selectivity curve. Gel filtration media are supplied with information about selectivity, as shown for Superdex in Figure 1.5. The curve is a plot of the partition coefficient K av against the log of the molecular weight for a set of standard proteins (for calculation of K av , see Chapter 7 Gel filtration in theory).

18-1022-18 AK 150.25

0.50

0.751.00K av

logarithmic scale

M r 1010101010106

Fig.1.5..Selectivity curves for Superdex.

Selectivity curves are almost linear in the range K av = 0.1 to K av = 0.7 and can be used to determine the fractionation range of a gel filtration medium (Fig 1.6).

1.0

0.7

0.1

K av

Fig.1.6..Defining fractionation range and exclusion limit from a selectivity curve.

The fractionation range defines the range of molecular weights that have partial access to the pores of the matrix; that is molecules within this range should be separable by high resolution fractionation. The exclusion limit for a gel filtration medium, also determined from the selectivity curve, indicates the size of the molecules that are excluded from the pores of the matrix and therefore elute in the void volume.

The steeper the selectivity curve, the higher the resolution that can be achieved.

When choosing a medium, consider two main factors:

1. The aim of the experiment (high resolution fractionation or group separation).

2. The molecular weights of the target proteins and contaminants to be separated.

16 18-1022-18 AK The final scale of purification should also be considered. Figure 1.7 gives some guidance to media selection. All media are available in prepacked columns, which is recommended if you have little experience in column packing.10M 2r 103104105106107108Superdex 30 prep grade

Superdex 75 prep grade

Superdex 200 prep grade Superdex Peptide

Superdex 75

Superdex 200

Superose 6 prep grade Superose 12 prep grade Superose?6Superose 12Sephacryl? S-100 HR Sephacryl S-200 HR Sephacryl S-300 HR Sephacryl S-400 HR Sephacryl S-500 HR Sephacryl S-1000 SF Sephadex G-10Sephadex G-25 SF Sephadex G-25F Sephadex G-25M Sephadex G-50F Exclusion limit Exclusion limit

Exclusion limit Sephadex LH-20

H i g h r e s o l u t i o n f r a c t i o n a t i o n

G r o u p s e p a r a t i o n /D e s a l t i n g Resolution Fig.1.7..Gel filtration media fractionation range guide.

Superdex is the first choice for high resolution, short run times, and high recovery. Superdex prep grade and Sephacryl are suitable for fast, high recovery separations at laboratory and industrial scale.

Superdex, Sephacryl, or Superose are high resolution media with a wide variety of fractionation ranges. In cases when two media have similar fractionation ranges, select the medium with the steepest selectivity curve (see chapter 2, 3, and 4 for the respective medium) for the best resolution of all the sample components. If a specific component is of interest, select the

medium where the log of molecular weight for the target component falls in the middle of the selectivity curve.

Sephadex is recommended for rapid group separations such as desalting and buffer

exchange. Sephadex is used at laboratory and production scale, before, between or

after other chromatography purification steps.

For group separations, select gel filtration media that elute high molecular weight molecules at the void volume to minimize peak broadening or dilution and reduce time in the column. The lowest molecular weight substances should appear by the time one column volume of buffer has passed through the column.

Table.1.2. Sephadex media properties

Medium Cut-off Application.examples

Sephadex G-10700Desalting of peptides

Sephadex G-251500Desalting of proteins and oligonucleotides

Sephadex G-505000Removal of free labels from labeled macromolecules Sample and buffer preparation

Removal of particles in the sample is extremely important for gel filtration. Clarifying a sample before applying it to a column will avoid the risk of blockage, reduce the need for stringent washing procedures and extend the life of the medium.

Samples must be clear and free from particulate matter, especially when working with bead sizes of 34 μm or less.

Appendix 3 contains an overview of sample preparation techniques. For small sample volumes a syringe-tip filter of cellulose acetate or PVDF can be sufficient. 5e0e3db65022aaea988f0f2dposition

The pH, ionic strength and composition of the sample buffer will not significantly affect resolution as long as these parameters do not alter the size or stability of the proteins to be separated and are not outside the stability range of the gel filtration medium. The sample does not have to be in exactly the same buffer as that used to equilibrate and run through the column. Sample is exchanged into the running buffer during the separation, an added benefit of gel filtration.

Sample.concentration.and.viscosity

Gel filtration is independent of sample mass and hence sample concentration, as can be seen in Figure 1.8. High resolution can be maintained despite high sample concentration and, with the appropriate medium, high flow rates.

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18 18-1022-18 AK Column:

XK 16/70 (140 ml)Medium:

Superdex 200 prep grade Sample:

Solution of transferrin (M r 81 000) and IgG (M r 160 000) by equal weight Sample.volume:

0.8% × V t Buffer:

0.05 sodium phosphate, 0.1 M sodium chloride, pH 7.2Flow.rate:

1 ml/min (30 cm/h)

packed bed

0.0R e s o l u t i o n , R S

0.5

1.0

1.5Fig.1.8..Influence of sample concentration on the resolution of transferrin and IgG on Superdex 200 prep grade.

The solubility or the viscosity of the sample may however limit the concentration that can be used.

A critical variable is the viscosity of the sample relative to the running buffer, as shown by the change in elution profiles of hemoglobin and NaCl at different sample viscosities in Figure 1.9.High sample viscosity causes instability of the separation and an irregular flow pattern. This leads to very broad and skewed peaks, and the back pressure might increase.

Elution volume

Fig.1.9..Deteriorating separation caused by increasing viscosity. Elution diagrams obtained when hemoglobin (blue) and NaCl (red) were separated. Experimental conditions were identical except that the viscosities were altered by the addition of increasing amounts of dextran. Note that lowering flow rate will not improve the separation.

Samples should generally not exceed 70 mg/ml protein. Dilute viscous samples, but not

more than necessary to keep the sample volume low. Remember that viscosity varies

with temperature.

Sample.volume

Sample volume is one of the most important parameters in gel filtration. Refer to page 12 for more information.

5e0e3db65022aaea988f0f2dposition.

Buffer composition will generally not directly influence the resolution unless the buffer affects the shape or biological activity of the molecules. Extremes of pH and ionic strength and the presence of denaturing agents or detergents can cause conformational changes, dissociation or association of protein complexes.

Select buffer conditions that are compatible with protein stability and activity. The product

of interest will be collected in this buffer. Use a buffer concentration that maintains buffering capacity and constant pH. Use from 25 mM up to 150 mM NaCl to avoid nonspecific ionic interactions with the matrix which can be seen as delays in peak elution. Note that some proteins may precipitate in low ionic strength solutions. Volatile buffers such as ammonium acetate, or ammonium bicarbonate should be used if the separated product will be lyophilized.

Use high quality water and chemicals. Solutions should be filtered through 0.45 μm

or 0.22 μm filters before use. It is essential to degas buffers before any gel filtration

separation since air bubbles can significantly affect performance. Buffers will be

automatically degassed if they are filtered under vacuum.

When working with a new sample, try these conditions first: 0.05 M sodium phosphate,

0.15 M NaCl, pH 7.0 or select the buffer into which the product should be eluted for the

next step (e.g., further purification, analysis, or storage).

Avoid extreme changes in pH or other conditions that may cause inactivation or even

precipitation. If the sample precipitates in the gel filtration column, the column will be

blocked, possibly irreversibly, and the sample may be lost.

Denaturing.(chaotropic).agents.and.detergents

Denaturing agents such as guanidine hydrochloride or urea can be used for initial solubilization of a sample as well as in gel filtration buffers to maintain solubility. However, since the proteins will denature, chaotropics should be avoided unless denaturation is specifically desired.

Superdex and Sephacryl are in general more suitable than classical media such as Sepharose? or Sephadex for working under dissociating or denaturing conditions or at extreme pH values.

Detergents are useful as solubilizing agents for proteins with low aqueous solubility, such as membrane components, and will not affect the separation. Sometimes, denaturing agents

or detergents are necessary to maintain the solubility of the sample. Such additives must be present all the time, both in the running buffer and the sample buffer.

If high concentrations of additives are needed, use lower flow rates to avoid excessive

pressure since they may increase the viscosity of the buffer.

If proteins precipitate, elute later than expected, or are poorly resolved during gel filtration,

it is recommended to add a suitable concentration of a denaturing agent or detergent to the running buffer.

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