Determination of Binding Constants by Affinity Chromatography Method (CAT#: STEM-CT-3391-CJ)

In general affinity chromatography is composed of a stationary phase (solid phase) and a mobile phase. The mobile phase is the cell lysate or any mixture that contains biomolecules. A ligand that binds the target molecule is attached covalently to the solid phase. The interaction between the solid and the mobile phase are exploited by affinity chromatography to get your desired substance in a pure form. The target molecule binds to the ligand, whereas most of the other molecules flow through. The target biomolecule is eluted by changing conditions (pH or salt concentrations) or by competition with a free ligand.The most important property which the solid phase should have is ligand immobilization. Various materials like acrylates or silica gels are appropriate. To prevent steric interference of the target molecule to the ligand, an inhibitor is attached to the solid phase. This inhibitor is known as the spacer.

Biochemistry

1. Preparation of Column: The column is loaded with solid support such as sepharose, agarose, cellulose etc.. Ligand is selected according to the desired isolate. Spacer arm is attached between the ligand and solid support.
2. Loading of Sample: Solution containing a mixture of substances is poured into the elution column and allowed to run at a controlled rate.
3. Elution of Ligand-Molecule Complex: Target substance is recovered by changing conditions to favor elution of the bound molecules.

• Sample: Plants; Natural Food; Protein; Drug; Pollutants; Blood; Saliva; Serum; Plasma; Antibodies; Viruses & More
• Equipment: Agarose; Silica gel; Aluminium oxide; Acrylate; Organic polymers; Wash Buffer
• (Optional): Ligand; Spacer arm; Column

1. Affinity chromatography is one of the most useful methods for the separation and purification of specific products.
2. It is essentially a sample purification technique, used primarily for biological molecules such as proteins.
3. Affinity chromatography can also be used to remove specific contaminants, such as proteases.
4. Limited availability and high cost of immobilized ligands.
5. Proteins get denatured if required pH is not adjusted.