Polyethylene Glycol (PEG) and Pegylation of Proteins

Polyethylene glycol is a typically biologically inert, non-immunogenic chemical that confers greater water solubility to proteins, labeling tags and crosslinkers with which it is incorporated as constituent chemical group. This page provides a brief introduction to the use and types of PEG reagents available to protein biology researchers.

Page Contents:

• Introduction
• Crosslinkers with PEG spacers
• PEG-containing Biotinylation Reagents
• PEG-containing Fluorescent Labeling Reagents
• Pegylation Reagents for Protein and Surface Modification

Introduction

What is PEG? (PEG Nomenclature)

PEG is the common abbreviation for polyethylene glycol – or, more properly, poly(ethylene glycol) – which refers to a chemical compound composed of repeating ethylene glycol units:

Depending on how one chooses to define the constituent monomer or parent molecule (as ethylene glycol, ethylene oxide or oxyethylene), PEG compounds are also known as PEO (polyethylene oxide) and POE (polyoxyethylene):

Purified PEG is most commonly available commercially as mixtures of different oligomer sizes in broadly or narrowly defined molecular weight (MW) ranges. For example, "PEG 600" typically denotes a preparation that includes a mixture of oligomers having an average MW of 600. Likewise, "PEG 10000" denotes a mixture of PEG molecules (n = 195 to 265) having an average MW of 10,000g/mol.

Properties of PEG

Poly(ethylene glycol) has several chemical properties that make it especially useful in various biological, chemical and pharmaceutical settings:

• Non-toxic and non-immunogenic – can be added to media and attached to surfaces and conjugated to molecules without interfering with cellular functions or target immunogenicities
• Hydrophilic (aqueous-soluble) – attachment to proteins and other biomolecules decreases aggregation and increases solubility
• Highly flexible – provides for surface treatment or bioconjugation without steric hindrance

Defined-length PEG Reagents

Precise and versatile application of PEG in proteomics and other biological research methods depends upon the availability of polyethylene glycol derivatives of defined length (MW) that are activated with specific functional groups. Thermo Scientific Pierce Protein Research Products include a diverse array of such PEG-containing reagents. These Pierce Products provide the building blocks required to covalently attach or modify surfaces, proteins and other molecules with PEG-containing derivatives, a process called PEGylation. By contrast with traditional PEG compounds, Pierce PEGylation Reagents are composed of precisely-defined numbers of PEG units. The remainder of this page describes the various classes of Pierce PEGylation Reagents.

Crosslinkers with PEG spacers

Many kinds of Pierce Crosslinkers are available for protein, peptide and other macromolecular immobilization and conjugation needs. Both homobifunctional (identical reactive groups at either end) and heterobifunctional (different reactive groups at either end) crosslinkers are offered with a variety of spacer-arm lengths, solubility and cleaving characteristics.

The wide selection of crosslinking reagents now includes those that contain discrete-length polyethylene glycol spacers. These PEG groups increase reagent and conjugate solubility, minimize toxic and immunological effects compared to non-PEG spacers, and provide several options for accommodating specific crosslinking distances.

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Crosslinking and Protein Modification

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Crosslinking Reagents

Amine-reactive Pegylated Crosslinkers

The BS(PEG)n reagents are homobifunctional, amine-to-amine crosslinkers that contain N-hydroxy-succinimide (NHS) esters at both ends of a PEG spacer spacer.

An amine-reactive crosslinker that contains a PEG spacer arm.

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BS(PEG)5
BS(PEG)9

Sulfhydryl-reactive Pegylated Crosslinkers

BM(PEG)n reagents are homobifunctional, sulfhydryl-reactive crosslinkers that contain the maleimide group at either end of a PEG spacer.

A sulfhydryl-reactive crosslinker that contains a PEG spacer arm.

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BM(PEG)2
BM(PEG)3

Bifunctional Pegylated Crosslinkers

The SM(PEG)n reagents are amine-to-sulfhydryl linkers that contain an NHS ester at one end and a maleimide group at the other. SM(PEG)n crosslinkers have the same heterobifunctional structure as the popular reagent SMCC (Part No. 22360 or 22322). Six varieties with 2 to 24 PEG units provide spacer lengths from 18 to 95 angstroms.

Amine-to-sulfhydryl crosslinkers that contain a PEG spacer arm.

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SM(PEG)2
SM(PEG)4
SM(PEG)6
SM(PEG)8
SM(PEG)12
SM(PEG)24

PEG-containing Biotinylation Reagents

Labeling antibodies and other molecules with biotin is the basis for many assay and purification platforms used in all areas of proteomics and molecular biology research. Thermo Scientific Pierce EZ-Link Biotinylation Reagents have many different target reactivities, spacer arm lengths, and solubility and cleavability characteristics.

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Biotinylation

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Biotin Labeling Reagents

Amine-reactive Pegylated Biotinylation Reagents

The wide selection of biotin-labeling reagents now include several classes of compounds that incorporate discrete-length polyethylene glycol groups as the primary constituent of the spacer arm. These PEG groups increase reagent and conjugate solubility and minimize toxic and immunological effects compared to non-PEG spacers. The alternative spacer lengths enable optimization of conjugate function for specific biotin-binding assays involving streptavidin, avidin or NeutrAvidin Protein.

NHS-PEG4-Biotin is the PEG equivalent of the popular Sulfo-NHS-LC-Biotin (Part No. 21335) and related reagents. Although the PEG analogs do not use the water-soluble Sulfo-NHS form of the amine-reactive group, equivalent reagent water solubility and membrane impermeability are conferred by the hydrophilic polyethylene glycol spacer arm. In addition, experiments have demonstrated that antibodies labeled with PEG-containing biotin tags retain better solubility (less aggregation during long-term storage) than those labeled using non-PEG counterparts.

An amine-reactive biotinylation reagent with a PEG spacer arm.

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NHS-PEG4-Biotin

NHS-PEG12-Biotin

NHS-SS-PEG4-Biotin
(cleavable)

TFP-PEG3-Biotin
(tetrafluorophenyl)

Sulfhydryl-reactive Pegylated Biotinylation Reagents

A sulfhydryl-reactive biotinylation reagent with a PEG spacer arm.

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Maleimide-PEG2-Biotin

Maleimide-PEG11-Biotin

Iodoacetyl-PEG2-Biotin

Other Pegylated Biotinylation Reagents

A carbohydrate-reactive biotinylation reagent with a PEG spacer arm.

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Hydrazide-PEG4-Biotin
Alkoxyamine-PEG4-Biotin
Alkoxyamine-PEG12-Biotin
Alkoxyamine-PEG8-SS-Biotin
(all carbohydrate reactive)

TFPA-PEG3-Biotin
(nonspecific)

Psoralen-PEG3-Biotin
(DNA intercalating)

PEG-containing Fluorescent Labeling Reagents

The use of fluorescent probes for various detection methods continues to expand. New types and derivatives of fluorescent compounds continue to be developed and introduced for use in research methods. This expansion includes the incorporation of PEG groups in fluorescent labeling compounds to increase their solubility and stability for various applications, including cellular and in vivo imaging.

Typical fluorescent labeling reactions require the dye to first be dissolved in anhydrous dimethyl formamide (DMF) or another suitable organic solvent before adding a specific molar amount of dye to an amine-free buffer containing the protein to be labeled. However, the higher solubility of pegylated fluors permits protein solutions to be added directly to the labeling medium.

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Fluorescent Probes

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Fluorescent Labeling Reagents

DyLight 680-4xPEG NHS Ester
DyLight 680-4xPEG Maleimide

Pegylation Reagents for Protein and Surface Modification

Certain experimental systems and assay platforms depend on the ability to alter the mass, solubility or other properties of proteins, immunogens, reaction vessels and other materials. PEGylation, the addition of ethylene glycol or ethylene oxide polymers, is a useful method of making these modifications.

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Pegylation Reagents

Amine-reactive Pegylation Reagents

Covalent modification with PEG groups requires PEG compounds that contain a reactive or targetable functional group at one end. The simplest method to pegylate proteins, which are rich in surface primary amines, is to use a PEG compound that contains an NHS ester group at one end. This is the basis for the MS(PEG)n reagents, which are available in four discrete PEG lengths (n = 4, 8, 12 and 24).

Amine-reactive pegylation reagents.

TMS(PEG)n is a branched form of this reagent, containing three methyl-PEG12 arms attached to a PEG4-NHS ester stem.

SAT(PEG)4 contains the amine-reactive NHS-ester group at one end and a protected sulfhydryl group (S-acetyl) at the other end. Like its non-PEG analog, SATA (Part No. 26102), SAT(PEG)4 allows the conversion of primary amines to sulfhydryl groups by addition of short spacer arms. The reagent is most often used as part of a crosslinking or immobilization strategy.

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MS(PEG)4
MS(PEG)8
MS(PEG)12
MS(PEG)24
   Methyl-PEG-NHS

TMS(PEG)12
   Tri(methyl-PEG)-NHS

SAT(PEG)4
   Thiol-PEG-NHS

Sulfhydryl-reactive Pegylation Reagents

MM(PEG)n and TMM(PEG)n are linear and branched reagents for pegylating sulfhydryl groups. At the end opposite the inert methyl group(s) is a maleimide moiety, which reacts to form stable thioether bonds with sulfhydryl groups. MM(PEG)n is available in four PEG lengths (n = 12 and 24). TMM(PEG)n contains three methyl-PEG12 arms that branch from a PEG4-maleimide stem.

Sulfhydryl-reactive pegylation reagents.

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MM(PEG)12
MM(PEG)24
   Methyl-PEG-Malemide

TMM(PEG)n
   Tri(methyl-PEG)-Maleimide

Pegylated Amino Acids and Amine Compounds

MA(PEG)n and CA(PEG)n are polyethylene glycol compounds of discrete length (n = 4, 8, 12 and 24) that contain methyl-and-amine or carboxyl-and-amine ends. While these functional groups are not spontaneously reactive, they are easily targeted by various crosslinking and immobilization reagents for construction of surface chemistries and other uses.

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CA(PEG)4
CA(PEG)8
CA(PEG)12
CA(PEG)24
   Carboxyl-PEG-Amine

MA(PEG)4
MA(PEG)8
MA(PEG)12
MA(PEG)24
   Methyl-PEG-Amine

Pegylated Carboxy and Thiol Compounds

Thiol-containing pegylation compounds.

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CT(PEG)12
   Carboxyl-PEG-Thiol

MT(PEG)4
   Methyl-PEG-Thiol

ML(PEG)4
   Methyl-PEG-Lipoamide