Single-molecule photobleaching (smPB) technique is a powerful tool for characterizing molecular assemblies. It canprovide a direct measure of the number of monomers constituting a given oligomeric particle and generate the oligomer sizedistribution in a specimen. A major current application of this technique is in understanding protein aggregation, which islinked to many incurable diseases. Quantitative measurement of the size distribution of an aggregating protein in aphysiological solution remains a difficult task, since techniques such as dynamic light scattering or fluorescence correlationspectroscopy (FCS) can provide an average size, but cannot accurately resolve the underlying size distribution. Here wedescribe the smPB method as implemented on a home-built total internal reflection fluorescence microscope (TIRF). Wefirst describe the construction of a TIRF microscope, and then demonstrate the power of smPB by characterizing a solutionof Amylin (hIAPP) oligomers, a 37-residue peptide whose aggregation is associated with Type II diabetes. We compare ourresults with FCS data obtained from the same specimen, and discuss the advantages and disadvantages of the twotechniques.
Volume 46, 2020
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