Download Cromatografía de afinidad

Principle of Affinity Chromatography
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
Materials
- The matrix
- The ligand
- Spacer Arms
- Coupling gel
- Group specific adsorbents (type - specificity)
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 polyvalen
metal ions
6x HIS-TAGGED PROTEIN PURIFICATION
WITH PDC USING PDC KIT
Conclusion
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).