Burst analysis spectroscopy: A versatile single-particle approach for studying distributions of protein aggregates and fluorescent assemblies

Jason Puchalla, Kelly Krantz, Robert Austin, Hays Rye

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Many essential cellular functions depend on the assembly and disassembly of macromolecular complexes. The size, form, and distribution of these assemblies can be heterogeneous and complex, rendering their detailed characterization difficult. Here we describe a simple non-correlation-based method capable of directly measuring population distributions at very low sample concentrations. Specifically, we exploit the highest signal-to-noise light bursts from single fluorescent particles transiting a confocal excitation spot to recursively determine the brightness and size distribution of complex mixtures of fluorescent objects. We refer to this method as burst analysis spectroscopy (BAS) and demonstrate the sensitivity of this technique by examining the free-solution, time-resolved distribution of assembled protein aggregates by using two fluorescently labeled proteins: the aggregation-prone, chaperonin-dependent, folding model protein ribulose-bisphosphate carboxylase/oxygenase (RuBisCO), and an amyloidogenic fragment of the yeast prion protein Sup35. We find that the assembly kinetics of both proteins display complex multimodal behavior not readily quantifiable with other methods.

Original languageEnglish (US)
Pages (from-to)14400-14405
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number38
DOIs
StatePublished - Sep 23 2008

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • Aggregation
  • Fluorescence spectroscopy
  • Microfluidics.

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