This study describes a method for the fast and effective affinity purification of sequence-specific DNA binding proteins. The method involves the synthesis of complementary oligodeoxynucleotides that contain a recognition site for the target protein, which are then annealed, 5'-phosphorylated, and ligated to form oligomers. These oligomers are covalently coupled to Sepharose CL-2B with cyanogen bromide to create an affinity resin. A partially purified protein fraction is combined with competitor DNA and passed through the DNA-Sepharose resin. The desired protein preferentially binds to the recognition sites in the resin, allowing for its purification. For example, Sp1 can be purified 500- to 1000-fold with 30% yield to an estimated 90% homogeneity using two sequential affinity chromatography steps. The method also allows for the simultaneous purification of multiple DNA binding proteins from the same extract using tandem affinity columns with different binding sites. Sequence-specific DNA binding proteins are crucial for many cellular processes, including transcription, replication, and recombination. However, these proteins are typically present in very low abundance, making their purification challenging. The method described here provides a means to purify these proteins effectively. The study also highlights the importance of using competitor DNA in the purification process to ensure that nonspecific proteins do not contaminate the target protein. The method has been successfully applied to purify Sp1, CAAT-binding transcription factor (CTF), and activator protein 1 (AP-1). The results show that the method is effective for purifying low abundance sequence-specific DNA binding proteins. The study also discusses the importance of buffer composition, competitor DNA concentration, and other factors in the success of the purification process. The method is applicable for the purification of other sequence-specific DNA binding proteins.This study describes a method for the fast and effective affinity purification of sequence-specific DNA binding proteins. The method involves the synthesis of complementary oligodeoxynucleotides that contain a recognition site for the target protein, which are then annealed, 5'-phosphorylated, and ligated to form oligomers. These oligomers are covalently coupled to Sepharose CL-2B with cyanogen bromide to create an affinity resin. A partially purified protein fraction is combined with competitor DNA and passed through the DNA-Sepharose resin. The desired protein preferentially binds to the recognition sites in the resin, allowing for its purification. For example, Sp1 can be purified 500- to 1000-fold with 30% yield to an estimated 90% homogeneity using two sequential affinity chromatography steps. The method also allows for the simultaneous purification of multiple DNA binding proteins from the same extract using tandem affinity columns with different binding sites. Sequence-specific DNA binding proteins are crucial for many cellular processes, including transcription, replication, and recombination. However, these proteins are typically present in very low abundance, making their purification challenging. The method described here provides a means to purify these proteins effectively. The study also highlights the importance of using competitor DNA in the purification process to ensure that nonspecific proteins do not contaminate the target protein. The method has been successfully applied to purify Sp1, CAAT-binding transcription factor (CTF), and activator protein 1 (AP-1). The results show that the method is effective for purifying low abundance sequence-specific DNA binding proteins. The study also discusses the importance of buffer composition, competitor DNA concentration, and other factors in the success of the purification process. The method is applicable for the purification of other sequence-specific DNA binding proteins.