Analysis of protein-protein interactions with a multi-capillary electrophoresis instrument.
Protein-protein interactions were analyzed by zone electrophoresis of premixed equilibrium mixtures of a fluorescence-labeled protein at a constant concentration and unlabeled protein at a variety of concentrations using a 96-CE instrument equipped with a LIF detector. The interactions between labeled-con A versus succinylated ovalbumin, labeled-trypsin versus four proteinaceous trypsin inhibitors and labeled-insulin versus seven anti-insulin monoclonal antibodies were analyzed using a dual buffer system, in which a 60 mM borate-Na buffer (pH 9.35) was used as electrophoresis buffer and 60 mM MOPS-Na (pH 7.35) containing 0.1% Tween 20 was used as a sample buffer. The dual buffer system allowed fast and reproducible analyses of interactions at a physiological pH using uncoated fused-silica capillaries. The change in the mobility moment, the first statistical moment of an electropherogram on the mobility axis (Shimura, K., Uchiyama, N., Enomoto, M., Matsumoto, H., Kasai, K., Anal. Chem. 2005, 77, 564-572), of the labeled proteins were analyzed as a function of the concentration of unlabeled proteins. The dissociation constants for seven antibodies ranging from sub nanomolar to micromolar was determined based on the results of one cycle of parallel electrophoresis runs, which completed in 30 min using 20 pmol (120 ng) of labeled insulin and 5 pmol (750 ng) each of the mAb.
Shimura
Kiyohito
K
Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo, Japan. shimura@icl.t.u-tokyo.ac.jp
Waki
Takuma
T
Okada
Masaki
M
Toda
Toshifusa
T
Kimoto
Izumi
I
Kasai
Ken-ichi
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002021:Buffers; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D007328:Insulin / Q000737:chemistry / Q000276:immunology; D046912:Multiprotein Complexes; D010047:Ovalbumin / Q000737:chemistry; D011480:Protease Inhibitors / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics; D014357:Trypsin / Q000737:chemistry
D000911:Antibodies, Monoclonal; D002021:Buffers; D007328:Insulin; D046912:Multiprotein Complexes; D011480:Protease Inhibitors; D011506:Proteins; D010047:Ovalbumin; D014357:Trypsin