Catching and separating protein ligands by functional affinity electrophoresis.
A new kind of affinity electrophoresis called functional affinity electrophoresis (FAEP) is a technique used to separate and/or capture proteins according to their functions in a native polyacrylamide gel. Protein A:immunoglobulin G, avidin:biotin, antibody:antigen, and concanavalin A:glycoprotein interactions are used to demonstrate this technique. Protein A, avidin, monoclonal anti-bovine serum albumin (BSA) antibody, and concanavalin A are embedded in distinct regions of a 7.5% native polyacrylamide gel. Some of each of the embedded proteins get covalently and/or noncovalently incorporated into the gel matrix network. Under electrophoresis conditions, these proteins do not show significant electrophoretic mobility or they migrate in a direction opposite to the protein analytes, as in avidin. We clearly observe that polyclonal anti-human myoglobin antibody, biotinylated insulin, BSA, and ovalbumin (glycoprotein) are captured and separated in distinct regions of a FAEP gel by protein A, avidin, monoclonal anti-BSA antibody, and concanavalin A, respectively.
Protein Research Laboratory, Research Resources Center, University of Illinois, Chicago, IL 60612, USA. boblee@uic.edu
Bao-Shiang
BS
Lee
Shalini
S
Gupta
Sangeeth
S
Krisnanchettier
Syed Salman
SS
Lateef
D000911:Antibodies, Monoclonal / Q000737:chemistry; D001360:Avidin / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D003208:Concanavalin A / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008024:Ligands; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D013205:Staphylococcal Protein A / Q000737:chemistry
D000911:Antibodies, Monoclonal; D008024:Ligands; D011506:Proteins; D013205:Staphylococcal Protein A; D003208:Concanavalin A; D001360:Avidin