Purification of the Glycosylated Beta Subunit of (Na,K)-ATPase by Lectin Affinity Chromatography (CAT#: STEM-ACM-0008-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.

Biochemical

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. Due to the ability of all lectins to identify and bind certain types of carbohydrate residues, this approach can be used to separate them.
2. The most common lectins employed in affinity columns are concanavalin A, soybean lectin, and wheat germ agglutinin.
3. Bioaffinity chromatography types can also utilise enzymes, inhibitors, cofactors, nucleic acids, hormones, and cell chromatography as ligands.