Preparation of affinity columns

Summary

This section will focus on the solid-phase technique of ligand solidification on agarose gels activated with cyanogen bromide. The vast majority of ligands, such as peptides, proteins, lectins, and nucleic acids, can be coupled relatively easily by this method. Source: Handbook of Protein Technology

Operation method

Ligand solidification technology

Principle

Cyanogen bromide-activated agarose gels for ligand solidification are one of the most commonly used solidification techniques in the laboratory. The principle is that the cyanogen bromide-activated medium reacts with the main nitrogen group of the ligand to form an isourea bond. However, a small amount of ligand will be dislodged when the coupled column is reused for many times, so other activation media should be considered for the reused column. The formation of the isourea bond is accompanied by an increase in the number of charged groups, which will affect the ion exchange. To overcome this effect, a buffered saline solution of at least 0.1 mol/L is used in the purification. This solid-phase method is simple, inexpensive, and widely used for proteins, nucleic acids, and lectins.

Materials and Instruments

Cyanogen bromide activated agarose gel
Hydrochloric acid Coupling buffer Blocking buffer
Glass funnel Glass rod Vacuum pump

Move

1. 2 g of cyanogen bromide-activated agarose gel is added to 1 mmol/L hydrochloric acid in a glass funnel and gently stirred with a glass rod for 20 min until the medium is completely dissolved. Let the suspension settle and remove the liquid with a vacuum pump. 1 g of dry gel gives 3.5 ml of solubilized gel. 2. Use 200 ml of 1 mmol/L agarose gel with 1 mmol/L hydrochloric acid as the solubilizer;

2. Wash the medium with 200 ml of 1 mmol/L hydrochloric acid three times and remove the liquid after each wash. 3;

3. wash the medium once with coupling buffer (0.1 mol/L sodium bicarbonate, 0.5 mol/L sodium chloride, pH 8.3). This step should be performed as quickly as possible, preferably within a few minutes, because the activating groups are easily hydrolyzed at the pH of the coupling buffer;

4. Transfer the activated agarose gel to a 30 ml flask of coupling buffer containing 60-400 nmol of ligand. Always take a small amount of the ligand solution to be measured if the binding efficiency is to be determined;

5. Mix gently, shake or vibrate for 2 h at room temperature or overnight at 4 °C. It is not advisable to use a magnetic stirrer as it can break up the agarose gel particles. If the binding efficiency is to be determined, take a small amount of the solution at the end of this step and leave it for measurement. 6;

6. wash the coupling medium once with 200 ml of coupling buffer in a sintered glass funnel. 7. transfer the coupling medium to a glass funnel;

7. Transfer the coupling medium to a flask containing 100 ml of blocking buffer (1 mol/L ethanolamine, pH 8.0 with hydrochloric acid). Incubate at room temperature for 2 h or overnight at 4°C. 8;

8. Wash the coupling medium once with 100 ml of coupling buffer and once with 100 ml of acetate buffer (0.1 mol/L sodium acetate, 0.5 mol/L sodium chloride, pH 4.0) in a sintered glass funnel;

9. Repeat step 8 four times;

10. if the coupling medium is not intended for immediate use, wash again with coupling buffer containing 0.02% sodium azide. 11. the ligand solidification has been completed;

11. Solidification of the ligand is complete and the coupled media is ready to be loaded onto the column.

Caveat

1. Estimation of binding efficiency. By detecting the ligand concentration in the solution to be tested before and after coupling, the position of the solidified ligand can be known, and thus the binding efficiency can be calculated. Generally speaking, 70% to 80% of the binding is optimal; low binding will lead to a decrease in the binding capacity of the affinity column; too much binding will produce spatial barriers resulting in a decrease in the binding capacity of the affinity column.

2. Many factors can affect the coupling, resulting in very low coupling efficiency. Common factors include: low ligand concentration, unsuitable pH, impure ligand, improper media handling, unsuitable coupling buffer, and inaccessible ligand. Among them, the ligand is not easy to approach often due to the existence of space barriers or ligand is too large. This may be improved by the addition of an appropriate amount of a nonionic descaler or a solubilizer. In addition, buffers containing amino groups such as Tris should not be used because of the possibility of coupling to the medium.

3. If the ligand is unstable at an alkaline pH, coupling can be performed in a lower pH buffer, but with reduced efficiency.

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