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Related Experiment Videos

DNA affinity chromatography.

P S Chockalingam1, L A Jurado, H W Jarrett

  • 1Department of Biochemistry, University of Tennessee, 858 Madison Avenue, Memphis, TN 38163, USA.

Molecular Biotechnology
|December 1, 2001
PubMed
Summary
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DNA-affinity chromatography purifies DNA-binding proteins using improved coupling methods. This technique enhances cellular process control by enabling the isolation of key proteins like transcription factors and polymerases.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Chromatography

Background:

  • DNA-affinity chromatography is crucial for purifying DNA-binding proteins that regulate cellular functions.
  • Advancements in coupling methodologies and support materials have improved its efficacy over time.

Purpose of the Study:

  • To describe optimized procedures for coupling oligonucleotides to solid supports for DNA-affinity chromatography.
  • To detail methods for quantifying oligonucleotide coupling and synthesizing affinity columns using enzymatic approaches.

Main Methods:

  • Coupling of 5'-aminoethyl-(dT)18 to N-hydroxysuccinimide/carbodiimide-activated silica.
  • Cyanogen bromide-mediated coupling of aminoethyl-(dT)18 to Sepharose.
  • Quantification of (dT)18 coupling via hybridization with a complementary (dA)18-labeled oligonucleotide.

Related Experiment Videos

  • Enzymatic synthesis of double-stranded DNA-silica or Sepharose affinity columns using DNA/RNA templates.

    • Main Results:

    • Established protocols for efficient coupling of DNA ligands to silica and Sepharose supports.
    • Demonstrated a reliable method for assessing the density of immobilized DNA ligands.
    • Showcased template-directed enzymatic synthesis as a base-modification-free approach for affinity column creation.

    Conclusions:

    • DNA-affinity chromatography is a versatile and powerful technique for protein purification.
    • The described methods offer improved strategies for developing highly specific affinity supports.
    • This technology is being extended for the purification of critical enzymes such as transcription factors, polymerases, and nucleases.