affinity chromatography

Summary

The affinity molecules with special structure are made into solid-phase adsorbent and placed in the chromatography column, when the protein mixture to be separated passes through the column, the proteins with affinity ability with the adsorbent will be adsorbed and retained in the chromatography column. Those proteins with no affinity will not be adsorbed and flow out directly, thus separating them from the separated proteins, and then selecting the appropriate eluent and changing the binding conditions to elute the bound proteins, this method of separating and purifying proteins is called affinity chromatography.

Operation method

Isolation of soluble or membrane-bound antigens

Materials and Instruments

Protein
Tris TSA lysis buffer Sodium deoxycholate Wash buffer Triethanolamine buffer
Chromatography columns Centrifuge tubes Centrifuge

Move

1. Prepare a Sepharose pre-column (5 ml column bed) quenched by activation and an Ab-Spharose immunoaffinity chromatography column and connect them in series.

2. Resuspend 50 g of cells in ice-cold TSA solution to ^1~5×108 cells/ml, or add 1~5 times the volume of TSA to the collected cell or tissue homogenate, and add an equal volume of ice-cold lysis buffer, and agitate for 1 h at 4℃.

3. Centrifuge at 4000 g for 10 min, remove the nuclei, gently pour out the supernatant and retain it.4. For purification of membrane antigens, add 0.2 times the volume of 5% sodium deoxycholate to the supernatant from the nuclei, place in a water bath or at 4°C for 10 min, transfer to a quick-seal centrifuge tube and centrifuge at 100,000 g for 1 h. Carefully remove the supernatant and retain it.5. Connect the precolumn to the immunoaffinity chromatography column.
Figure 1. Immunoaffinity chromatography column
6. Wash the column sequentially with the following buffers:
10 times the wash buffer of the column bed
Tris buffer, pH 8.0, 5 times the column bed
Tris buffer for 5x column bed, pH 9.0
Triethanolamine solution in 5x column beds
Wash buffer for 5x column bed

7. Spike the supernatant (reserving some sample for analysis) onto the pre-column and allow it to flow through the pre-column and the immunoaffinity chromatography column at a flow rate of 5 column beds/h. Collect the permeate in fractions of 1/10-1/100 of the volume of spiked supernatant.
8. Wash the columns with 5 times the volume of Wash Buffer and close the stopcock on both columns. Disconnect the affinity column from the pre-column and open the stopcock of the affinity column to allow the liquid on top of the column bed to flow to the surface of the column bed.

9. Wash the columns sequentially with the following solutions and collect the effluent fractions in sections:
5x volume of wash buffer
5x volume of Tris buffer, pH 8.0
5x volume of Tris buffer, pH 9.0

10. elute with 5x volume of Triethanolamine solution and collect each column bed volume of eluate in a test tube containing 0.2 volume of 1 mol/l Tris-Cl buffer, pH 6.7 to neutralize the spiked solution.11. Wash the column with 5x volume of TSA solution.12. Analyze antigen-containing eluate fractions: 50 μl of each eluate was analyzed by SDS-PAGE silver staining. Take 0.5~1 ml of the upper column sample and a representative penetrating effluent and eluate to be immunoprecipitated with Ab-Sepharose and followed by SDS-PAGE and silver-staining assay to determine if the column is saturated.

Caveat

At each buffer change, wash the immunoaffinity column as follows:1. close the stopcock and open the cap at the top end of the injection.

2. Using a syringe attached to the outlet of the tubing from the buffer reservoir, aspirate all liquid.

3. transfer the tube to another column wash buffer in another reservoir, aspirate with a syringe to fill the tube with liquid, remove the syringe, adjust the flow rate, and use this buffer to wash the inside of the column.

4. Open the stop valve, let the column buffer flow to the level of the bed, relax the upper cap of the column to let the buffer flow down to the level of a few centimeters above the bed of the column, tighten the cap, and start washing or elution.

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