In an anion exhcnage chromotography the bound protein is eluted by
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Elution with salt gradient. Addition of salt increases the number of ions competing with proteins for functional groups on the stationary phase. Proteins spend more time in the solution, the rate of their movement down the column increases dramatically, and proteins begin to elute from the column, usually in order of increasing charge. Most proteins are eluted at NaCl concentrations < 1M.
Elution by pH change. Change of pH in the column can be aimed to decrease the net absolute value of the charges of adsorbed proteins, decrease their attraction to the stationary phase, and accelerate the elution. In practice, pH changes in the column are difficult to control, as they do not reliably correspond to pH changes of the applied eluting buffer. This happens because of the buffering power of proteins adsorbed to the column and, for weak ion exchangers (see below), buffering power of the adsorbent functional groups themselves. Resolution of proteins by pH elution is achieved in a separate technique called “Chromatofocusing.”
Elution by affinity. Affinity elution can be achieved for a specific protein if and only if an oppositely charged ligand that will strongly bind to this protein is known and available. Addition of such a ligand to the eluting buffer will produce a protein+ligand species with a smaller absolute value of the net charge, and therefore the targeted protein will bind less to the stationary phase. Affinity elution is often useful in enzyme purifications
Elution by pH change. Change of pH in the column can be aimed to decrease the net absolute value of the charges of adsorbed proteins, decrease their attraction to the stationary phase, and accelerate the elution. In practice, pH changes in the column are difficult to control, as they do not reliably correspond to pH changes of the applied eluting buffer. This happens because of the buffering power of proteins adsorbed to the column and, for weak ion exchangers (see below), buffering power of the adsorbent functional groups themselves. Resolution of proteins by pH elution is achieved in a separate technique called “Chromatofocusing.”
Elution by affinity. Affinity elution can be achieved for a specific protein if and only if an oppositely charged ligand that will strongly bind to this protein is known and available. Addition of such a ligand to the eluting buffer will produce a protein+ligand species with a smaller absolute value of the net charge, and therefore the targeted protein will bind less to the stationary phase. Affinity elution is often useful in enzyme purifications
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