Free-solution, label-free molecular interactions studied by back-scattering interferometry.
Free-solution, label-free molecular interactions were investigated with back-scattering interferometry in a simple optical train composed of a helium-neon laser, a microfluidic channel, and a position sensor. Molecular binding interactions between proteins, ions and protein, and small molecules and protein, were determined with high dynamic range dissociation constants (Kd spanning six decades) and unmatched sensitivity (picomolar Kd's and detection limits of 10,000s of molecules). With this technique, equilibrium dissociation constants were quantified for protein A and immunoglobulin G, interleukin-2 with its monoclonal antibody, and calmodulin with calcium ion Ca2+, a small molecule inhibitor, the protein calcineurin, and the M13 peptide. The high sensitivity of back-scattering interferometry and small volumes of microfluidics allowed the entire calmodulin assay to be performed with 200 picomoles of solute.
Bornhop
Darryl J
DJ
Department of Chemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University, VU Station B 351822, Nashville, TN 37235-1822, USA. darryl.bornhop@vanderbilt.edu
Latham
Joey C
JC
Kussrow
Amanda
A
Markov
Dmitry A
DA
Jones
Richard D
RD
Sørensen
Henrik S
HS
D000595:Amino Acid Sequence; D000818:Animals; D019703:Calcineurin / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry; D004129:Dimethylpolysiloxanes; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007368:Interferometry / Q000379:methods*; D007700:Kinetics; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011817:Rabbits; D012031:Refractometry; D012828:Silicones; D012996:Solutions
D002147:Calmodulin; D004129:Dimethylpolysiloxanes; D007074:Immunoglobulin G; C107660:M13 protein (myosin light-chain kinase); D010446:Peptide Fragments; D012828:Silicones; D012996:Solutions; C013830:baysilon; D009219:Myosin-Light-Chain Kinase; D019703:Calcineurin; D002118:Calcium