Detection of small differences in actomyosin function using actin labeled with different phalloidin conjugates

This study shows that there is only a negligible difference in actomyosin function in the in vitro motility assay among actin filaments labeled with Rhodamine phalloidin (RhPh), Alexa-488 phalloidin (APh), and biotin-XX phalloidin (BPh). Similar results were obtained at varying ionic strengths (0.02...

Full description

Saved in:
Bibliographic Details
Published in:Analytical biochemistry 2005-03, Vol.338 (2), p.224-236
Main Authors: Balaz, Martina, Månsson, Alf
Format: Article
Language:English
Subjects:
Actins - analysis
Actins - chemistry
Actomyosin - chemistry
Actomyosin - physiology
Alexa-488 phalloidin
Animals
Biotin-XX phalloidin
Bovine cardiac actin
Cattle
In vitro motility assay
Muscle Contraction - physiology
Nanotechnology
Phalloidine - analogs & derivatives
Phalloidine - analysis
Phalloidine - chemistry
Rabbit skeletal muscle actin
Rats
Rhodamine phalloidin
Staining and Labeling - methods
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:This study shows that there is only a negligible difference in actomyosin function in the in vitro motility assay among actin filaments labeled with Rhodamine phalloidin (RhPh), Alexa-488 phalloidin (APh), and biotin-XX phalloidin (BPh). Similar results were obtained at varying ionic strengths (0.02–0.13 M), in the presence of imidazole or 3-[ N-morpholino]propanesulfonic acid (MOPS) buffer, and at varying MgATP concentrations (0.1–3 mM). If RhPh- and APh-labeled filaments were studied in a given flow cell, there was minimal variability in sliding velocity between the fluorophores (standard deviation of 3% of the absolute sliding velocity). The variability was considerably smaller than that between flow cells, allowing us to use dual labeling of different actin types and then apply analysis of variance to detect minor functional differences between them. Using this method, we could statistically verify a 4% difference ( P < 0.001) in sliding velocity (3 mM Mg ATP) between cardiac and skeletal muscle actin. Suggested improvements of the method would readily allow the detection of even smaller differences. We discuss implications of the results for nanotechnological applications, understanding actomyosin function, and reducing experimental costs and the use of laboratory animals.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2004.12.012