High-Affinity Iodoacetyl Agarose

1. Buffer Preparation:

It is recommended to prepare the buffers using high-purity water and sterilize them by filtration through a 0.45 μm membrane after preparation.

2. Coupling of Thiol-Containing Samples:

(1) Sample Preparation: Dissolve the thiol-containing protein, peptide, or other biological ligand in coupling buffer to a final concentration of approximately 1 mg/mL.

(2) Column Packing: Rinse the bottom frit of the column with coupling buffer and ensure that no air bubbles remain on the frit. Close the bottom outlet and leave 1-2 cm of coupling buffer at the bottom of the column. Thoroughly resuspend the High-Affinity Iodoacetyl Agarose, transfer an appropriate amount into the column, and allow the resin to settle slowly at the bottom.

(3) Equilibration: Equilibrate the column with 3 column volumes of coupling buffer. Allow the buffer to flow slowly through the column at approximately 1 mL/min. Repeat this procedure at least twice.

(4) Coupling: Close the bottom cap, add the sample, and allow the solution to enter the packed bed. Close the top cap and place the column on a shaker. Incubate at room temperature for 30 min. After coupling, secure the column on a stand, remove the bottom cap, allow the solution to flow out, and let the resin settle at the bottom.

Note: Add approximately 1 mL of sample per 1 mL of agarose resin.

(5) Washing: Wash the column with 3 column volumes of coupling buffer at approximately 1 mL/min. Collect the flow-through, measure its OD₂₈₀ value, and compare it with the OD₂₈₀ value before coupling to evaluate coupling efficiency.

(6) Blocking: Close the bottom cap and add a volume of blocking buffer equal to the column volume. Close the top cap and place the column on a shaker. Incubate at room temperature for 30 min. After blocking, secure the column on a stand, remove the bottom cap, allow the solution to flow out, and let the resin settle at the bottom.

(7) Storage: If the column will be used immediately, proceed directly to the antibody purification procedure. If the column will not be used immediately, wash it with 3 column volumes of binding/wash buffer, add 1 column volume of storage buffer, and store at 2-8°C. Do not freeze.

3. Antibody Purification by Column Chromatography:

(1) Equilibration: Equilibrate the column with at least 5 column volumes of binding/wash buffer. Allow the buffer to flow slowly through the column and let the resin settle at the bottom.

(2) Antibody Binding: Add the antibody sample and allow it to flow slowly through the column at approximately 0.5 mL/min. Collect the flow-through. If necessary, reload or recirculate the sample to improve recovery.

(3) Washing: Wash the column with 30-50 column volumes of binding/wash buffer until the OD₂₈₀ absorbance reaches a stable baseline. Use a flow rate of approximately 1.5 mL/min and collect the flow-through.

(4) Elution: After washing, elute the column with 5-10 column volumes of elution buffer. Protein elution may be monitored by measuring OD₂₈₀. Use a flow rate of approximately 1 mL/min, collect the eluate, and immediately add neutralization buffer at approximately 1/10 of the eluate volume to adjust the pH.

(5) SDS-PAGE Analysis: Analyze the purified antibody samples, including the flow-through, wash, and elution fractions, together with the original sample by SDS-PAGE to evaluate purification performance.

4. Resin Regeneration and Storage:

Wash the resin with 5 column volumes of binding/wash buffer, add 1 column volume of storage buffer, and store at 2-8°C. Do not freeze.