Affinity chromatography

The chromatographic methods discussed above are all  dependent upon the  gross  physicochemical  properties  of  the  protein.  However,  the biological  activity  of the  protein  is  generally  more  subtle  and  depends upon  the  very  specific,  complementary,  steric  relationship  between  the active site and a substrate (or inhibitor),  or a binding site  and a ligand, as the  case  may  be.  Affinity  chromatography  exploits  this  biospecific relationship  between a protein  and a  ligand,  to  specifically  select  out  a desired protein from a crude mixture, essentially in a single step.

The specific ligand, which in the case of an enzyme may be a substrate or an inhibitor, is immobilized by conjugation to an insoluble matrix,  in a manner  which does  not  interfere  with  its  interaction  with  the  protein.

This  may  require the  use of a spacer arm,  which typically  consists  of  a chain of about 6-10  carbon  atoms.  An affinity  chromatography  resin is thus  comprised  of  three  parts,  i)  the  matrix,  which  is  similar  to  the matrices used for ion-exchange chromatography, ii) a spacer arm,  and iii) the ligand.  Matrix/spacer arm combinations  are commercially  available, since these  are universal reagents,  and simply  require the  addition  of an appropriate ligand.

The sample  solution  is passed through the  column and by its specific interaction with the immobilized ligand the protein of interest is  retained, while all other  proteins  pass straight through  the  column.  Subsequently, the  protein  can  be eluted by a change  in  either  the  pH  or  ionic  strength of the buffer or by addition of a free  competing  ligand,  or of a chaotrope. Because the protein is immobilized in a small volume of resin,  affinity columns are generally quite small.  Also, the volume  of solution  in  which the  protein  occurs  may  be  large  and  to  pass  this  volume  through  the

References

-Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .