Affinity chromatography

Affinity chromatography developed the high affinity, specific, noncovalent binding of a protein to another molecule the ligand.  At first the ligand is covalently attached to porous matrix and an inert like as Sepharose. The protein combination is then passed down a column containing the immobilized ligand. The protein of  interest  will  connect  to  the  ligand,  while  all  other  proteins  pass  straight by the column. After extensive washing of the column with buffer to erase  nonspeci?cally  bound  proteins  the bound  protein  is free  from the immobilized  ligand  either  through adding  soluble  ligand  that  competes  with the immobilized ligand for the protein or through shifting the properties of the buffer like changing  the salt concentration or pH.  If soluble  ligand  is used  to elute  the protein from the column extensive dialysis frequent then has to be used to erase the  small  ligand  from  the  larger  protein.  Because  this  method  exploits  the frequently unique,  speci?c,  binding  properties  of the protein  it is quite possible  to divide  the protein  from  a mixture  of hundreds  of other  proteins  in a single chromatographic  step.   Generally   employed   combinations   of   immobilized ligand  and  protein  to  be  puri?ed  used  in  affinity  chromatographic  systems involve an inhibitor to purify an enzyme, an antibody to purify its antigen, a hormone for example insulin  to purify  its receptor, and a lectin for example concanavalin  A to purify a glycoprotein. Advances in recombinant  DNA technology mean which proteins  can  be  engineered  with  specific  sequences  of  amino  acids  at  the  C- terminal  end  a  so-called  tag.  A recombinant   tagged protein can then be expressed in a suitable cell system and the af?nity of the tag for an immobilized antibody or other molecule exploited to purify the protein.

Figure: Affinity chromatography.  (a) Schematic diagram of affinity chromatography; (b) elution diagram indicating that nonspecific proteins that do not bind to the immobilized ligand pass straight through the column, while the speci?c protein binds to the immobilized ligand and is eluted from the column only on addition of soluble ligand.