Concatemers, tandem copies of DNA elements ligated together, are widely used for the DNA affinity chromatography of transcription factors. Purification of different transcription factors using discrete, concatemeric and T18:A18 tailed DNA affinity columns was studied. Columns having a discrete DNA sequence bound by cytidylic-adenylic-adenylic-thymidylic oligonucleotide (CAAT) enhancer binding protein (C/EBP) yields significantly more green fluorescent protein-C/EBP (GFP-C/EBP) fusion protein than a concatemeric DNA column made from five tandem repeats of the same DNA sequence. For lac repressor protein, the concatemeric and T18:A18 tailed columns show greater retention times than a discrete, untailed DNA affinity column. It was observed that the T18:A18 tailed column gives better resolution than either the discrete or concatemeric columns, of mixtures containing both lac repressor and GFP-C/EBP. Discrete concatemeric and T18:A18 tail columns all bound the Sp1 transcription factor and showed similar retention. The T18:A18 tailed column gives higher yield for Sp1 than the other columns. Our study shows concatemeric columns do not have any distinct advantage for the three different transcription factors we studied including Sp1, the original justification for the concatemeric approach.