Purification of Labeled Antibodies Using Size-Exclusion Chromatography
Abstract
Many antibody labeling procedures call for a desalting or purification step requiring size-exclusion chromatography (SEC). The method outlined here contains information needed to desalt an antibody conjugate. Similar procedures would be used for ion-exchange chromatography using a gradient of increasing ionic strength. Resins can be purchased in bulk (as in this protocol), or commercially available columns are available.
MATERIALS
Reagents
DiH2O
Labeled antibody solution
Running buffer: PBS, Tris, HEPES, MES (type and pH will depend on the application)
Size-exclusion gel matrix (e.g., Sephadex; choice dependent on size of conjugate; see Table 1)
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Table 1 provides a list of SEC Sephadex resins and the effective separation range of each. Typically manufacturers give a volume/milliliter value. Your volume of sample should be no more than 5% of the column volume for desalting and no more than 2% of the column volume when separating molecules that are close in size.
SEC Sephadex resins and the effective separation range of each
Equipment
Chromatography equipment (optional)
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Prepoured columns are available, but even common laboratory materials (i.e., 10-mL syringe with filter paper or frit) can be adapted for use.
Glass or polypropylene column
Glass or polypropylene tubes
Glassware (beaker or flask)
Peristaltic pump (optional)
Spectrophotometer
METHOD
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1. Swell the resin in 200–300 mL of DiH2O overnight at room temperature.
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Some resins require different swelling conditions including degassing. Refer to the manufacturer's instructions.
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2. Decant the liquid without losing the swelled gel.
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3. Wash the gel twice with DiH2O, letting the matrix settle and decanting the liquid.
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4. Replace the DiH2O with running buffer. Let the gel settle, decant, and add fresh running buffer. Repeat this again.
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5. Pour the swelled gel into a column and allow it to settle using either gravity or a pump.
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6. When adding additional matrix, gently stir the interface with a sterile pipette.
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Keeping the poured resin as continuous as possible will ensure the best separation.
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7. Equilibrate the column with either 10 column volumes of buffer, or if the buffer was replaced during the swelling process (Step 4), then two column volumes will be sufficient.
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8. Expose the gel bed and gently load the sample onto the gel bed, minimizing disturbance of the surface.
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9. Allow the sample to completely enter the gel bed, and then begin to gently add running buffer to the gel bed.
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10. Collect 0.5-mL fractions from the column.
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11. After several milliliters of running buffer have entered the gel bed, additional buffer can be added to the top of the gel.
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12. Measure the OD of the fractions in a spectrophotometer. The first peak is the antibody and should begin to elute in approximately the sixth fraction. Pool the main peak and measure the OD again to determine the antibody concentration.
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See Troubleshooting.
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13. Store the antibody conjugate as described above and in Introduction: Antibody Purification and Storage (Berg and Fishman 2019).
TROUBLESHOOTING
Problem (Step 12): The protein elutes too quickly (crack in column).
Solution: Collect the sample, concentrate, repour the column, and repeat the protocol.
Problem (Step 12): The protein elutes too slowly (wrong sizing matrix chosen).
Solution: Use a different matrix that separates in a smaller MW range.
Footnotes
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From the Antibodies collection, edited by Edward A. Greenfield.
