On-column capture of a specific protein separated by SDS-CGE using an immunological reaction on magnetic beads.
The on-column capture of a specific protein using magnetic beads was applied to SDS-CGE. In a preliminary study, an immunological reaction in the presence of SDS, using a batch method, was attempted. Carbonic anhydrase (CA), alpha-lactalbumin (LA), and HSA were denatured by heating in the presence of SDS and 2-mercaptoethanol, and then reacted with anti-CA that had been immobilized on magnetic beads. Not only native CA, but also the denatured CA reacted with anti-CA, even in the presence of SDS. Therefore, the on-column capture of denatured CA separated by SDS-CGE was attempted using a two-point detection technique. A mixture of proteins, containing LA, CA, and HSA, were separated by SDS-CGE according to their Mr. The CA was then specifically captured on anti-CA-immobilized magnetic beads, which were packed between two detection windows in the capillary column, during the electrophoresis. The results show that the technique leads to information similar to that obtained by Western blotting, i.e., a protein can be identified by its Mr and reaction with its antibody.
Kaneta
Takashi
T
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Fukuoka, Japan.
Takahashi
Masanori
M
Imasaka
Totaro
T
D000906:Antibodies / Q000302:isolation & purification; D002256:Carbonic Anhydrases / Q000276:immunology / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D008280:Magnetics; D008863:Microspheres; D011489:Protein Denaturation / Q000187:drug effects; D012967:Sodium Dodecyl Sulfate
D000906:Antibodies; D012967:Sodium Dodecyl Sulfate; D002256:Carbonic Anhydrases