Substance Isolated by Affinity Chromatography Ligand Enzyme Substrate, cofactor Antibody Antigen, Virus, Cell Polysaccharide, glycoprotein Lectin Nucleic.

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Presentation transcript:

Substance Isolated by Affinity Chromatography Ligand Enzyme Substrate, cofactor Antibody Antigen, Virus, Cell Polysaccharide, glycoprotein Lectin Nucleic acid binding protein Nucleic Acid Hormone receptor Hormone Principle of Affinity Chromatography

- The matrix - The ligand - Spacer Arms - Coupling gel - Group specific adsorbents (type - specificity) Materials

Tipos especiales de cromatografía de afinidad *Cromatografía de afinidad sobre metales inmobilizados IMAC - MCAC *Cromatografía de afinidad por unión covalente Empleo de matrices con tiopropilo o glutatión *Cromatografía de afinidad de células Emleo de matrices con ligandos que pueden interactuar con receptores de membrana o Ac unidos a la superficie celular.

Immobilized metal ion affinity chromatography (IMAC) or Metal chelate affinity chromatography (MCAC) for 6x His-tagged protein and Zn-protein purification in a single step and for first attempts at general purification of proteins (see PDC and PDC kit). PDC The best chelator for protein purification in IMAC It is known in IMAC (Immobilized Metal ion Affinity Chromatography) that 1. Immobilized IDA (Iminodiacetic acid) is a tridentate ligand at physiological pH 2. Immobilized NTA (Nitrilotriacetic acid) 3. Immobilized PDC (Pentadentate chelator) Iminodiacetic acid-Agarose for IMAC PDC (pentadentate chelator) & PDC kit for IMAC PMIB (polyvalent metal ion binding resin) for water, buffers & proteins free of polyvalent metal ions

6x HIS-TAGGED PROTEIN PURIFICATION WITH PDC USING PDC KIT Zn-PDC allowed the purification of HSP 60 in a single step with a recovery of 15 mg of protein per ml of wet gel (Panel C, lane 5 & 6). Conclusion