Femtomole mixer for microsecond kinetic studies of protein folding.
We have developed a microfluidic mixer for studying protein folding and other reactions with a mixing time of 8 mus and sample consumption of femtomoles. This device enables us to access conformational changes under conditions far from equilibrium and at previously inaccessible time scales. In this paper, we discuss the design and optimization of the mixer using modeling of convective diffusion phenomena and a characterization of the mixer performance using microparticle image velocimetry, dye quenching, and Forster resonance energy-transfer (FRET) measurements of single-stranded DNA. We also demonstrate the feasibility of measuring fast protein folding kinetics using FRET with acyl-CoA binding protein.
Hertzog
David E
DE
Mechanical Engineering Department, Stanford University, Stanford, California 94305, USA.
Michalet
Xavier
X
Jäger
Marcus
M
Kong
Xiangxu
X
Santiago
Juan G
JG
Weiss
Shimon
S
Bakajin
Olgica
O
D000214:Acyl Coenzyme A / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000032:analysis; D004268:DNA-Binding Proteins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007700:Kinetics; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding
D000214:Acyl Coenzyme A; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins