DNA-Binding Protein Purification by Affinity Chromatography (CAT#: STEM-MB-1287-LGZ)

Affinity chromatography relies on the specific and reversible binding of proteins to matrix-bound ligands. Ligands can bind directly to the protein of interest or to a tag covalently attached to the protein. Affinity chromatography is generally the most reliable purification procedure and is often used in the early stages of a purification scheme. This specific affinity interaction enables the capture of the target while simultaneously removing contaminants or other molecules in solution and enriching or purifying the target molecule away from all other molecules that cannot bind the ligand in a single step.

For the purification of DNA-binding protein.

1. In affinity chromatography, proteins are loaded onto a column under conditions that affect the binding between the protein (or tag) and its ligand.
2. Wash the bound protein under conditions that do not disrupt specific interactions, but can disrupt any nonspecific interactions between contaminating proteins and the stationary phase.
3. The bound protein is then eluted with a buffer containing competing molecules or conditions that disrupt all protein/protein interactions.
4. Competing molecules bind to the ligand, displacing the target protein. This competing molecule is usually removed from the protein of interest by another chromatographic procedure or dialysis.

• Sample containing DNA-binding protein