Immobilized Metal ion Affinity Chromatography (IMAC)

  • IMAC is a powerful and versatile protein purification technique used to purify proteins, peptides, and other biomolecules that have an affinity for metal ions. Proteins containing amino acids like histidine, cysteine, or tryptophan bind to metal ions due to their inherent affinity.
  • As a type of affinity chromatography, IMAC operates on the principle that immobilized metal ions on a solid support attract and bind to target protein and peptide molecules. The target molecule can then be eluted from the column by changing the pH or increasing the ionic strength of the buffer, or by adding agents like EDTA or imidazole.
  • Metal ions, such as Ni²⁺, Co²⁺, Cu²⁺, Zn²⁺, and Fe³⁺, are immobilized onto a chromatographic medium using chelating agents like iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA).
  • Advantages: 
    • High selectivity
    • High capacity: IMAC resins can bind large amounts of protein.  
    • Versatility: Variety of proteins including antibodies, enzymes, and recombinant proteins can be purified by IMAC.
    • Ease of use
  • Limitations:
    • Metal ion leaching
    • Non-specific binding
  • Applications:
    • Purification of proteins from a complex mixture of biomolecules (e.g., Lactate dehydrogenase)
    • Purification of enzymes from cell lysates and other sources
    • Purification of antibodies from serum and other sources
    • Purification of recombinant proteins (e.g., purification of His-tagged proteins)
    • Phosphoprotein Fractionation: IMAC can be used in conjunction with mass spectrometry for the fractionation of phosphoproteins
    • Biomarker Detection: It can be used for the pretreatment of samples for the detection of biomarkers in serum, urine, and tissues

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