SulfoLink Coupling Resin is a pre-activated resin that can be used to immobilize antigens by reacting with sulfhydryl or amino groups to purify antibodies from immune serum.

Purification process

SulfoLink Coupling Resin has different coupling conditions for sulfhydryl and amino groups. Please refer to process 1.1 for coupling antigens containing sulfhydryl groups and process 1.2 for coupling antigens containing amino groups.

1.1 Coupling process of sulfhydryl antigens

1.1.1 Buffer preparation

It is recommended to filter the water and Buffer with 0.22μm or 0.45μm filter membrane before use.

Coupling solution: 50 mMTris, 5 mMEDTA-Na, pH 8.5

Sealing solution: 50 mMTris, 5 mMEDTA-Na, 50 mML cysteine, pH 8.5

Protective solution: 20 mM phosphate buffer, 20% ethanol, pH 8.0

Binding/cleaning solution: 20 mM phosphate buffer, pH 8.0

Eluent: 100 mM glycine, pH 2.5-3.0

Neutralizing solution: 1 M Tris-HCl, pH 8.5

1.1.2 Antigen preparation

Always ensure that the sulfhydryl group of the antigen sample is in the reduced state before use (Ellman's Reagent can be used to test the content of free sulfhydryl group). If the sulfhydryl group has been oxidized, it is necessary to reduce the antigen, generally recommended to use the reducing agent TCEP (Tris(2-carboxyethyl) phosphine), TCEP solution is very stable, can selectively and efficiently open the disulfide bond in the antigen, and does not affect the coupling reaction between the antigen and the resin. The amount of TCEP added per milligram of antigen does not exceed 12mg, which needs to be optimized by itself. If DTT and other reducing agents containing sulfhydryl groups are used, the reducing agent must be removed after the sample is treated, otherwise the coupling efficiency of antigen and resin will be affected. The conjugate solution was used to dissolve the antigen and a final concentration of 1-3 mg/ml was prepared. It is recommended to use the solution on the spot, storage time is too long will affect the coupling effect.

1.1.3 Antigen coupling

1) Take appropriate amount of SulfoLink Coupling Resin and add it into a suitable gravity column. Drain the protective fluid by gravity and balance the resin with coupling fluid of 3 times the volume of the column. When the coupling fluid is drained, add the coupling fluid of 3 times the volume of the column. A total of 9 times the column volume of coupling fluid is used.

2) Close the lower outlet of the column, add an equal volume of sulfhydryl antigens, mix, take out in a suitable centrifuge tube, and incubate at 28℃for 30min.

Note: Ensure that the resin is fully suspended, otherwise the coupling efficiency will be greatly affected.

3) Remove the above reaction system, transfer it to the gravity column, drain the solution, collect the outflow, and then clean the resin with the coupling liquid of 3 times the volume of the column, and combine the two outflows.

Note: If necessary, you can use Ellman's Reagent to test the sulfhydryl content in it to get the residual antigen, so as to calculate the coupling efficiency.

4) Close the lower outlet of the column, add an equal volume of sealing liquid, mix well, transfer to a suitable centrifuge tube, and incubate at 28℃for 30min.

5) Remove the reaction system, transfer it to the gravity column, and drain the sealing liquid therein.

Note: If used immediately, you can refer to 1.1.4 operations. If used later, the resin can be cleaned with a binding liquid of 3 times the column volume, and then stored in a protective liquid of the same volume at 2℃-8℃.

1.1.4 Antibody purification

1) The resin coupled with the antigen is loaded into a suitable chromatographic column, and the binding solution is balanced with 5 times the column volume, so that the filler is in an environment that is easier to bind with the antibody, on the one hand, to protect the target antibody, and on the other hand, to improve the antibody binding efficiency.

2) Add the sample containing antibodies to the balanced resin. In order to ensure full contact between the antibody and the resin and improve the recovery rate of the target antibody, the loading flow rate can be controlled at 0.5-1 ml/min, and the effluent can be collected.

3) Wash with 10-15 times the column volume of the detergent to remove the impurity protein unless specifically adsorbed, improve the purity of the target antibody, and collect the detergent.

4) Using 5-10 times the column volume of eluent, collect the elution component, that is, the target antibody.

Note: In order to maintain antibody activity, the elution component needs to be immediately dialyzed to a pH 7.0-8.0 buffer, or a neutralizing solution with 1/10 times the volume of the elution component is added first, the elution component is neutralized, and then dialyzed.

5) The binding liquid of 3 times the column volume and the deionized water balance resin of 5 times the column volume are used successively, and finally the protective liquid of 5 times the column volume is balanced, and then stored in the protective liquid of the same volume and stored at 2℃-8℃to prevent the filler from being contaminated by bacteria.

1.2 Coupling process containing amino antigen

1.2.1 Buffer preparation

It is recommended to filter the water and Buffer with 0.22μm or 0.45μm filter membrane before use.

Coupling solution: 0.1MNaHCO3, 0.5MNaCl, pH 8.5

Sealing solution: 50 mMTris, 5 mMEDTA-Na, 50 mML cysteine, pH 8.5

Protective solution: 20 mM phosphate buffer, 20% ethanol, pH 8.0

Binding/cleaning solution: 20 mM phosphate buffer, pH 8.0

Eluent: 100 mM glycine, pH 2.5-3.0

Neutralizing solution: 1 M Tris-HCl, pH 8.5

1.2.2 Antigen preparation

Amino group is a relatively stable group, so there is no special requirement for antigens containing amino group. The antigen was dissolved in the coupling solution and a final concentration of 5-10 mg/ml was prepared.

1.2.3 Antigen coupling

1) Take appropriate amount of SulfoLink Coupling Resin and add it into a suitable gravity column. Drain the protective fluid by gravity and balance the resin with coupling fluid of 3 times the volume of the column. When the coupling fluid is drained, add the coupling fluid of 3 times the volume of the column. A total of 9 times the column volume of coupling fluid is used.

2) Close the lower outlet of the column, add an equal volume of amino containing antigen, mix, remove and transfer to a suitable centrifuge tube, incubate at 28℃for 3-5 hours, or incubate at 2-8℃for overnight (12-15 hours).

Note: Ensure that the resin is fully suspended, otherwise the coupling efficiency will be greatly affected.

3) Remove the above reaction system, transfer it to the gravity column, and collect the antigenic solution, and then clean the resin with the coupling solution of 3 times the volume of the column, and combine two outflows, leaving for testing.

4) Close the lower outlet of the column, add an equal volume of sealing liquid, mix well, remove and transfer to a suitable centrifuge tube, and incubate at 28℃for 30min.

5) Remove the reaction system, transfer it to the gravity column, and drain the sealing liquid therein.

Note: If used immediately, you can refer to 1.2.4 operations. If used later, the resin can be cleaned with a binding liquid of 3 times the column volume, and then stored in a protective liquid of the same volume at 2℃-8℃.

1.2.4 Antibody purification

1) The resin coupled with the antigen is loaded into a suitable chromatographic column, and the binding solution is balanced with 5 times the column volume, so that the filler is in an environment that is easier to bind with the antibody, on the one hand, to protect the target antibody, and on the other hand, to improve the antibody binding efficiency.

2) Add the sample containing antibodies to the balanced resin. In order to ensure full contact between the antibody and the resin and improve the recovery rate of the target antibody, the loading flow rate can be controlled at 0.5-1 ml/min, and the effluent can be collected.

3) Wash with 10-15 times the column volume of the detergent to remove the impurity protein unless specifically adsorbed, improve the purity of the target antibody, and collect the detergent.

4) With 5-10 times the column volume of eluent, collect the elution component, that is, the target antibody.

Note: In order to maintain antibody activity, the elution component needs to be immediately dialyzed to a pH 7.0-8.0 buffer, or a neutralizing solution with 1/10 times the volume of the elution component is added first, the elution component is neutralized, and then dialyzed.

5) The binding liquid of 3 times the column volume and the deionized water balance resin of 5 times the column volume are used successively, and finally the protective liquid of 5 times the column volume is balanced, and then stored in the protective liquid of the same volume and stored at 2℃-8℃to prevent the filler from being contaminated by bacteria.

1.3 SDS-PAGE detection

The effluents, scrugs and elutes of purified antibody samples and the original antibody samples were tested by SDS-PAGE.

Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.