lambda-Repressor oligomerization kinetics at high concentrations using fluorescence correlation spectroscopy in zero-mode waveguides.
Fluorescence correlation spectroscopy (FCS) has demonstrated its utility for measuring transport properties and kinetics at low fluorophore concentrations. In this article, we demonstrate that simple optical nanostructures, known as zero-mode waveguides, can be used to significantly reduce the FCS observation volume. This, in turn, allows FCS to be applied to solutions with significantly higher fluorophore concentrations. We derive an empirical FCS model accounting for one-dimensional diffusion in a finite tube with a simple exponential observation profile. This technique is used to measure the oligomerization of the bacteriophage lambda repressor protein at micromolar concentrations. The results agree with previous studies utilizing conventional techniques. Additionally, we demonstrate that the zero-mode waveguides can be used to assay biological activity by measuring changes in diffusion constant as a result of ligand binding.
Samiee
K T
KT
School of Applied & Engineering Physics, Cornell University, Ithaca, New York 14853, USA.
Foquet
M
M
Guo
L
L
Cox
E C
EC
Craighead
H G
HG
D000465:Algorithms; D003460:Crystallization / Q000379:methods*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry*; D019281:Dimerization; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007700:Kinetics; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods*; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry; D012097:Repressor Proteins / Q000032:analysis* / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D014764:Viral Proteins / Q000032:analysis* / Q000737:chemistry*; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins