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HomeProtein PurificationSample Preparation for Size Exclusion Chromatography

Sample Preparation for Size Exclusion Chromatography

Samples for chromatographic purification should be clear and free from particulate matter. Simple steps to clarify a sample before beginning purification will avoid clogging the column, can reduce the need for stringent washing procedures, and can extend the life of the chromatographic medium.

Sample extraction procedures and the selection of buffers, additives, and detergents are determined largely by the source of the material, the stability of the target molecule, the chromatographic techniques that will be employed, and the intended use of the product. These subjects are dealt with in general terms in the Protein Purification Handbook and more specifically according to target molecule in the Recombinant Protein Handbook, and Antibody Purification Handbook, available from Cytiva.

Sample clarification

Centrifugation and filtration are standard laboratory techniques for sample clarification and are used routinely when handling small samples.

It is highly recommended to centrifuge and filter any sample immediately before chromatographic purification.

Centrifugation

Centrifugation removes lipids and particulate matter, such as cell debris. If the sample is still not clear after centrifugation, use filter paper or a 5 μm filter as a first step and one of the filters below as a second step filter.

  • For small sample volumes or proteins that adsorb to filters, centrifuge at 10 000 × g for 15 min.
  • For cell lysates, centrifuge at 40 000 to 50 000 × g for 30 min.
  • Serum samples can be filtered through glass wool after centrifugation to remove any remaining lipids.

Filtration

Filtration removes particulate matter. Membrane filters that give the least amount of nonspecific binding of proteins are composed of cellulose acetate or PVDF.

For sample preparation before chromatography, select a filter pore size in relation to the bead size of the chromatographic medium (Table A3.1).

Nominal pore size of filterParticle size of chromatography medium
1 μm90 μm and upwards
0.45 μm34 μm
0.22 μm3, 10, 15 μm or when extra clean samples or sterile filtration is required
Table A3.1Filter pore size recommendations

Check the recovery of the target protein in a test run. Some proteins can adsorb nonspecifically to filter surfaces.

Desalt

Denaturation

Denaturing agentTypical conditions for useRemoval/comment
Urea2 to 6 MRemove using Sephadex G-25
Guanidine hydrochloride3 to 6 MRemove using Sephadex G-25
Table A3.2Common denaturing agents

Details taken from: Scopes R.K., Protein Purification, Principles and Practice, Springer, (1994), J.C. Janson and L. Rydén, Protein Purification, Principles, High Resolution Methods and Applications, 2nd ed. Wiley Inc, (1998) and other sources.

Precipitation and resolubilization

Specific sample preparation steps might be required if the crude sample is known to contain contaminants such as lipids, lipoproteins, or phenol red, which can build up on a column. Sample preparation can also be required if certain gross impurities, such as bulk protein, need be removed before any chromatographic step.

Fractional precipitation is occasionally used at laboratory scale to remove gross impurities but is generally not required in purification of affinity-tagged proteins. In some cases, precipitation can be useful as a combined protein concentration and purification step.

Precipitation techniques separate fractions by the principle of differential solubility. For example, because protein species differ in their degree of hydrophobicity, increased salt concentrations can enhance hydrophobic interactions between the proteins and cause precipitation. Fractional precipitation can be applied to remove gross impurities in three different ways, as shown in Figure A3.1.

Three ways to use precipitaion

Figure A3.1.Three ways to use precipitaion.

Precipitation techniques can be affected by temperature, pH, and sample concentration.

These parameters must be controlled to ensure reproducible results.
Most precipitation techniques are not suitable for large-scale preparation.

Precipitation agentTypical conditions for useSample typeComment
Ammonium sulfateAs described below.> 1 mg/mL proteins, especially immunoglobulins.Stabilizes proteins, no denaturation; supernatant can go directly to HIC. Helps to reduce lipid content.
Dextran sulfateAdd 0.04 mL of 10% dextran sulfate and 1 mL of 1 M CaCl2 per mL of sample, mix 15 min, centrifuge at 10 000 × g, discard pellet.Samples with high levels of lipoprotein, e.g., ascites.Precipitates lipoprotein.
PolyvinylpyrrolidineAdd 3% (w/v), stir 4 h, centrifuge at 17 000 × g, discard pellet.Samples with high levels of lipoprotein, e.g., ascites.Alternative to dextran sulfate.
Polyethylene glycol (PEG, Mr > 4000)Up to 20% (w/v).Plasma proteins.No denaturation, supernatant goes directly to IEX or AC, complete removal can be difficult. Stabilizes proteins.
Acetone (cold)Up to 80% (v/v) at 0 °C.Collect pellet after centrifugation at full speed in microcentrifuge.Can denature protein irreversibly. Useful for peptide precipitation or concentration of sample for electrophoresis.
Polyethyleneimine0.1% (w/v). Precipitates aggregated nucleoproteins.
Protamine sulfate1% (w/v). Precipitates aggregated nucleoproteins.
Streptomycin sulfate1% (w/v). Precipitates nucleic acids.
Caprylic acid(X/15) g where X = volume of sample.Antibody concentration should be > 1 mg/mL.Precipitates bulk of proteins from sera or ascites, leaving immunoglobulins in solution.
Table A3.3Examples of precipitation techniques

Details taken from: Scopes R.K., Protein Purification, Principles and Practice, Springer, (1994), J.C. Janson and L. Rydén, Protein Purification, Principles, High Resolution Methods and Applications, 2nd ed. Wiley Inc, (1998).

Ammonium sulfate precipitation

Ammonium sulfate precipitation is frequently used for initial sample concentration and clean up. As the concentration of the salt is increased, proteins will begin to “salt out.” Different proteins salt out at different concentrations, a process that can be taken advantage of to remove contaminating proteins from the crude extract. The salt concentration needs to be optimized to remove contaminants and not the desired protein. An additional step with increased salt concentration should then precipitate the target protein. If the target protein cannot be safely precipitated and redissolved, only the first step should be employed. HIC is often an excellent next purification step, as the sample already contains a high salt concentration and can be applied directly to the HIC column with little or no additional preparation. The elevated salt level enhances the interaction between the hydrophobic components of the sample and the chromatography medium.

Solutions needed for precipitation:

  • Saturated ammonium sulfate solution (add 100 g ammonium sulfate to 100 mL distilled water, stir to dissolve).
  • 1 M Tris-HCl, pH 8.0.
  • Buffer for first purification step.

Some proteins can be damaged by ammonium sulfate. Take care when adding crystalline ammonium sulfate: high local concentrations can cause contamination of the precipitate with unwanted proteins.

For routine, reproducible purification, precipitation with ammonium sulfate should be avoided in favor of chromatography.

In general, precipitation is rarely effective for protein concentrations below 1 mg/mL.

  1. Filter (0.45 μm) or centrifuge the sample (10 000 × g at 4 °C).
  2. Add 1 part 1 M Tris-HCl, pH 8.0 to 10 parts sample volume to maintain pH.
  3. Stir gently. Add ammonium sulfate solution, drop by drop. Add up to 50% saturation*. Stir for 1 h.
  4. Centrifuge for 20 min at 10 000 × g.
  5. Remove supernatant. Wash the pellet twice by resuspension in an equal volume of ammonium sulfate solution of the same concentration (i.e., a solution that will not redissolve the precipitated protein or cause further precipitation). Centrifuge again.
  6. Dissolve the pellet in a small volume of the buffer to be used for the next step.
  7. Ammonium sulfate is removed during clarification/buffer exchange steps with Sephadex G-25 using desalting columns (see Chapter 5).

*The percentage saturation can be adjusted either to precipitate a target molecule or to precipitate contaminants.

The quantity of ammonium sulfate required to reach a given degree of saturation varies according to temperature. Table A3.4 shows the quantities required at 20 °C.

Final percent saturation to be obtained+A1:R23
 20253035404550556065707580859095100
Starting percentAmount of ammonium sulfate to add (grams) per liter of solution at 20 °C
0113144176208242277314351390430472516561608657708761
585115146179212246282319358397439481526572621671723
105786117149182216251287325364405447491537584634685
15285888119151185219255293331371413456501548596647
200295989121154188223260298337378421465511559609
25 0296091123157191228265304344386429475522571
30  0306192125160195232270309351393438485533
35   0306294128163199236275316358402447495
40    0316396130166202241281322365410457
45     0316498132169206245286329373419
50      0326599135172210250292335381
55       03366101138175215256298343
60        03367103140179219261305
65         03469105143183224267
70          03470107146186228
75           03572110149190
80            03673112152
85             03775114
90              03776
95               038
Table A3.4Quantities of ammonium sulfate required to reach given degrees of saturation at 20 °C

Removal of lipoproteins

Lipoproteins and other lipid material can rapidly clog chromatography columns and it is advisable to remove them before beginning purification. Precipitation agents such as dextran sulfate and polyvinylpyrrolidine, described under Fractional precipitation, are recommended to remove high levels of lipoproteins from samples such as ascites fluid.

Centrifuge samples to avoid the risk of nonspecific binding of the target molecule to a filter.

Samples such as serum can be filtered through glass wool to remove remaining lipids.

Materials
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