Affinity Chromatography AC

With affinity chromatography (AC), the biospecific interactions between, for example, a protein and the matching antibody are used to separate the protein or the antibody. This separation is based on a specific and reversible interaction between the molecules. A typical apphcation is the purification of monoclonal antibodies. Under the given conditions, they bond to a stationary phase that is covered with the protein A. After a washing step, the conditions are changed (often change in the pH value) such that the antibody elutes. 

AC is a highly selective separation method, which finds widespread application in research and production. 

Serum albumin will be eluted at the given conditions at about 24 min. To detect very similar conjugation products modify the gradient 0-10 min 0 30% B, 10-20 min 30 50% B, 30-40 min 50 80% B, 40-45 min 100% B, and 45-60 min 0% B. Monitor proteins at 225 nm or at the wavelength of the absorption maximum of the respective hapten. 

Run the respective gradient program from time to time without protein to check for impurities. Inject about 2 ml 0.1 N NaOH to remove residual protein and to clean the columns and tubings. 

The first step in affinity chromatography is mostly the preparation of the affinity matrix by covalent immobilization (coupling) 

Elution of the bound ligand can be done by competition with the free (unbound) ligand or by partial denaturation of the pro- 

Reagent suitable Proteins Nucleic -NH2 -SH Polysac- at pH for acids charides 

---

The classification of chromatography as gel permeation chromatography (GPC size-exclusion chromatography, SEC gel filtration, GF), ion exchange chromatography (lEC), hydrophobic interaction chromatography (HIC), and affinity chromatography (AC) is... 

Ion exchange chromatography, hydrophobic interaction chromatography (HlC), and affinity chromatography (AC) show some similarities concerning practical realization therefore, most of the hints given for lEC are applicable for HlC and AC. Examples for AC are given in Protocols 3.6.2.4 (biospecific desorption) and 3.6.2.5 (elution by partial denaturation). 

Affinity chromatography (AC) is a fractionation technique widely used in targeted proteomics or protein-protein interaction approaches. This method utilizes an interaction or affinity of a target protein to a substrate (or another protein) immobilized on a support matrix. Figure 17.4 demonstrates that AC can be incorporated into 3D proteomic approaches (Lee and Lee, 2004). Traditionally, AC has been used to purify, for example, carbohydratebinding proteins from Diplostomum pseudo-spathaceum (Mikes and Man, 2003) and the detoxification superfamily glutathione transferase (GST) from parasitic flatworms (Brophy and Barrett, 1990), specifically shown in... 

Affinity chromatography (AC) Ligand specificity High capacity High resolution + + + + + + + +... 

A variety of micropellicular packing materials has been developed for the analysis of both small and large molecules by various HPLC modes, including ion exchange (lEC), metal interaction (MIC), reversed phase (RPC) [4], and affinity chromatography (AC) [5]. Besides analytical applications, other possible utilization of micropellicular stationary phases includes fundamental kinetic and thermodynamic studies of the retention mechanisms on a well-defined surface. Nevertheless, a relatively limited variety of micropellicular... 

---