| Characterization of brightness and stoichiometry of bright particles by flow-fluorescence fluctuation spectroscopy. | |
| | |
MedLine Citation:
|
PMID: 21044607 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
|
Characterization of bright particles at low concentrations by fluorescence fluctuation spectroscopy (FFS) is challenging, because the event rate of particle detection is low and fluorescence background contributes significantly to the measured signal. It is straightforward to increase the event rate by flow, but the high background continues to be problematic for fluorescence correlation spectroscopy. Here, we characterize the use of photon-counting histogram analysis in the presence of flow. We demonstrate that a photon-counting histogram efficiently separates the particle signal from the background and faithfully determines the brightness and concentration of particles independent of flow speed, as long as undersampling is avoided. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used to determine stoichiometry of protein complexes in vivo and in vitro. We apply flow-FFS to determine the stoichiometry of the group specific antigen protein within viral-like particles of the human immunodeficiency virus type-1 from the brightness. Our results demonstrate that flow-FFS is a sensitive method for the characterization of complex macromolecular particles at low concentrations. |
| | |
Authors:
|
Jolene Johnson; Yan Chen; Joachim D Mueller |
Related Documents
:
|
8042507 - Transient leukemoid reaction in an extremely premature neonate. 9519227 - Blood eosinophil and monocyte counts are related to smoking and lung function. 15876797 - Counting absolute number of lymphocytes in quail whole blood by flow cytometry. 3999687 - Effects of ketamine anaesthesia, stress and repeated bleeding on the haematology of ver... 17955907 - Frequency and causes of infectious abortion in a dairy herd in queretaro, mexico. 17650777 - Estimating the cost of blood: past, present, and future directions. |
Publication Detail:
|
Type: Journal Article; Research Support, N.I.H., Extramural |
Journal Detail:
|
Title: Biophysical journal Volume: 99 ISSN: 1542-0086 ISO Abbreviation: Biophys. J. Publication Date: 2010 Nov |
Date Detail:
|
Created Date: 2010-11-03 Completed Date: 2011-01-28 Revised Date: 2011-11-03 |
Medline Journal Info:
|
Nlm Unique ID: 0370626 Medline TA: Biophys J Country: United States |
Other Details:
|
Languages: eng Pagination: 3084-92 Citation Subset: IM |
Copyright Information:
|
Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved. |
Affiliation:
|
School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, USA. |
Export Citation:
|
APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
|
Biophysical Phenomena Fluorescent Dyes HIV Antigens / chemistry HIV-1 / chemistry Humans Macromolecular Substances / chemistry Microspheres Particle Size Proteins / chemistry* Spectrometry, Fluorescence / methods* Virion / chemistry gag Gene Products, Human Immunodeficiency Virus / chemistry |
| Grant Support | |
ID/Acronym/Agency:
|
GM64589/GM/NIGMS NIH HHS; R01 GM064589-10/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
|
0/Fluorescent Dyes; 0/HIV Antigens; 0/Macromolecular Substances; 0/Proteins; 0/gag Gene Products, Human Immunodeficiency Virus |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
Previous Document: Mechanical forces regulate elastase activity and binding site availability in lung elastin.
Next Document: Cross-validating FRAP and FCS to quantify the impact of photobleaching on in vivo binding estimates.