Immunoaffinity purification of the calpains.
A monoclonal antibody to the small subunit common to both mu- and m-calpains can be used in an immunoaffinity column to purify either mu- or m-calpain in a proteolytically active form. Extracts in 150 mM NaCl, pH 7.5, are loaded onto a column containing the anti-28-kDa antibody; the column is washed with 500 mM NaCl, pH 7.5, and the bound calpain is eluted with 150 mM NaCl, 50 mM Tris-HCl, pH 9.5, and 1 mM EDTA. These elution conditions do not affect the proteolytic activity of either mu- or m-calpain. It is most efficient to reduce the volume and to remove any proteolytic activity from crude extracts by using successive phenyl Sepharose and ion-exchange columns before loading onto the immunoaffinity column. The column purifies m-calpain more effectively than mu-calpain; m-calpain is greater than 90% pure after a single pass through this column, whereas mu-calpain can be purified to >70% purity. The epitope for the monoclonal antibody is between amino acids 92 and 104 (numbers for human calpain) in the 28-kDa subunit. Evidently, this area is shielded in the calpain molecule in a way that affects binding of the antibody to the native molecule.
Cong
Jinyang
J
Muscle Biology Group, University of Arizona, Tucson, AZ 85721, USA.
Thompson
Valery F
VF
Goll
Darrel E
DE
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D002154:Calpain / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular
D000911:Antibodies, Monoclonal; D000939:Epitopes; D002154:Calpain; C449000:m-calpain; C449002:mu-calpain