AbstractDye affinity chromatography is a protein purification procedure based on the high affinity of immobilized dyes for the binding sites on many proteins. It is a rapid, inexpensive, and versatile method that is applicable to the purification of crude cellular extracts. This unit presents protocol for the three types of dye affinity chromatography: negative chromatography, positive chromatography, and tandem chromatography. An initial protocol describes a chromatographic procedure in which a small volume of the protein mixture to be purified is applied to a series of miniature columns, each containing a different immobilized dye. Analysis of the flowthrough and bound material allows determination of the optimum dye material for larger‐scale purification. An alternate procedure describes a similar initial selection procedure using centrifugation instead of chromatography. A support protocol describes a simple procedure for immobilization of free dyes.