Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

J Vis Exp. 2022 Apr 6:(182). doi: 10.3791/63459.

Abstract

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An important first step in any membrane-dependent reaction is binding of protein on the phospholipid membrane. An approach to studying protein interaction with lipid membranes has been developed using fluorescently labeled proteins and flow cytometry. This method allows the study of protein-membrane interactions using live cells and natural or artificial phospholipid vesicles. The advantage of this method is the simplicity and availability of reagents and equipment. In this method, proteins are labeled using fluorescent dyes. However, both self-made and commercially available, fluorescently labeled proteins can be used. After conjugation with a fluorescent dye, the proteins are incubated with a source of the phospholipid membrane (microvesicles or cells), and the samples are analyzed by flow cytometry. The obtained data can be used to calculate the kinetic constants and equilibrium Kd. In addition, it is possible to estimate the approximate number of protein binding sites on the phospholipid membrane using special calibration beads.

Publication types

  • Video-Audio Media
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell-Derived Microparticles* / metabolism
  • Flow Cytometry / methods
  • Fluorescent Dyes / chemistry
  • Humans
  • Membranes / metabolism
  • Phospholipids* / chemistry

Substances

  • Fluorescent Dyes
  • Phospholipids