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

Summary

Here, we describe a set of methods for characterizing the interaction of proteins with membranes of cells or microvesicles.

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 K_d. In addition, it is possible to estimate the approximate number of protein binding sites on the phospholipid membrane using special calibration beads.

Introduction

Biomembranes separate the inner contents of animal cells and extracellular space. Note that membranes also surround microvesicles formed during the cell's life cycle and organelles. The cell membrane is predominantly composed of lipids and proteins. Membrane proteins perform signaling, structural, transport, and adhesive functions. However, the lipid bilayer is also essential for the interrelation of the animal cell with the extracellular space. This paper proposes a method for studying the peripheral interaction of external proteins with the lipid membrane.

The most striking example of reactions occurring on the outer membrane layer of....

Protocol

1. Fluorescent protein labeling

1. Material preparation
   1. Prepare 1 M Sodium bicarbonate buffer, pH 9.0, store it at 4 °C, and use it within one week.
   2. Prepare 1.5 M hydroxylamine hydrochloride buffer, pH 8.5, immediately before use.
   3. Prepare a 10 mg/mL solution of fluorescent dye (see the Table of Materials) in dimethylsulfoxide.
      NOTE: This solution can be stored for a month at -20 °C in the dark.
   4. Prepare solutions of purified antibodies or other proteins at 1-10 mg/mL.
      NOTE: Avoid buffers containing ammonium ions or primary amines. Replace the buffers containing Tris or g....

Results

The flow cytometry method described herein is used to characterize the binding of plasma coagulation proteins to activated platelets. In addition, phospholipid vesicles PS:PC 20:80 were applied as a model system. This paper mainly focuses on artificial phospholipid vesicles as an example. The parameters of the cytometer, in particular, the photomultiplier tube (PMT) voltage and the compensation must be selected for each specific device, the object of study (cells, artificial or natural microvesicles), and the dyes used. .......

Discussion

The proposed method can be adapted for a rough characterization of the interaction of proteins with phospholipid membranes from various sources and compositions. The quantitative flow cytometry described here concedes to surface plasmon resonance (SPR) in several parameters. In particular, it has a lower sensitivity and time resolution and requires fluorescent labeling of proteins. Fluorescent labeling can lead to a change in conformation and loss of activity for many proteins and therefore requires careful control. Howe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
A23187	Sigma Aldrich	C7522-10MG
Alexa Fluor 647 NHS Ester (Succinimidyl Ester)	Thermo Fisher Scientific	A37573	fluorescent dye
Apyrase from potatoes	Sigma Aldrich	A2230
BD FACSCantoII	BD Bioscience
bovine serum albumin	VWR Life Science AMRESCO	Am-O332-0.1
Calcium chloride, anhydrous, powder, ≥97%	Sigma Aldrich	C4901-100G
Cary Eclipse Fluorescence Spectrometer	Agilent
D-(+)-Glucose	Sigma Aldrich	G7528-1KG
DiIC16(3) (1,1'-Dihexadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate)	Thermo Fisher Scientific	D384
DMSO	Sigma Aldrich	D8418
EDTA disodium salt	VWR Life Science AMRESCO	Am-O105B-0.1
FACSDiva	BD Bioscience		cytometry data acquisition software
FlowJo	Tree Star		cytometer software for data analysis
HEPES	Sigma Aldrich	H4034-500G
Human Factor X	Enzyme research	HFX 1010
Hydroxylamine hydrochloride	Panreac	141914.1209
L-α-phosphatidylcholine (Brain, Porcine)	Avanti Polar Lipids	840053P
L-α-phosphatidylserine (Brain, Porcine) (sodium salt)	Avanti Polar Lipids	840032P
Magnesium chloride	Sigma Aldrich	M8266-100G
Mini-Extruder	Avanti Polar Lipids	610020-1EA
OriginPro 8 SR4 v8.0951	OriginLab Corporation		Statistical software
Phosphate Buffered Saline (PBS) Tablets, Biotechnology Grade	VWR Life Science AMRESCO	97062-732
Potassium chloride	Sigma Aldrich	P9541-500G
Prostaglandin E1	Cayman Chemical	13010
Sephadex G25	GE Healthcare	GE17-0033-01	gel filtration medium for protein purification
Sepharose CL-2B	Sigma Aldrich	CL2B300-500ML	gel filtration medium for platelet purification
Sodium bicarbonate	Corning	61-065-RO
Sodium chloride	Sigma Aldrich	S3014-500G
Sodium phosphate monobasic	Sigma Aldrich	S3139-250G
Spin collumns with membrane 0.2 µm	Sartorius	VS0171
Trisodium citrate dihydrate	Sigma Aldrich	S1804-1KG