Polyethylene Glycol (PEG) and PEGylation of Proteins
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.
Synthesis of PEG
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.
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
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.
Crosslinking Reagents Technical Handbook
This 45-page guide is of value to the novice as well as those who have previous experience with crosslinking reagents. It begins with a basic discussion on crosslinking and the reagents that are used. The guide also contains a discussion on various applications where crosslinking has been applied, including the powerful label transfer technique for identifying or confirming protein interactions. Crosslinking chemistry is addressed in an easy-to-follow format designed to convey the important information you need without getting lost in details. Each Thermo Scientific Pierce crosslinking reagent is shown along with its structure, molecular weight, spacer arm length and chemical reactivity. The handbook concludes with a list of excellent references on crosslinker use and a glossary of common crosslinking terms.
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.
Bioconjugate Techniques, 3rd Edition
Bioconjugate Techniques, 3rd Edition (2013) by Greg T. Hermanson is a major update to a book that is widely recognized as the definitive reference guide in the field of bioconjugation.
Bioconjugate Techniques is a complete textbook and protocols-manual for life scientists wishing to learn and master biomolecular crosslinking, labeling and immobilization techniques that form the basis of many laboratory applications. The book is also an exhaustive and robust reference for researchers looking to develop novel conjugation strategies for entirely new applications. It also contains an extensive introduction to the field of bioconjugation, which covers all the major applications of the technology used in diverse scientific disciplines, as well as tips for designing the optimal bioconjugate for any purpose.
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. Thermo Scientific Pierce BS (PEG) 5 is a bis-succinimide ester-activated PEG compound used for crosslinking between primary amines (—NH2) in proteins and other molecules. The N-hydroxysuccinimide ester (NHS) groups at either end of the PEG5 spacer react specifically and efficiently with lysine and N-terminal amino groups at pH 7-9 to form stable amide bonds.
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. Thermo Scientific Pierce BM (PEG) 3 is a bismaleimide-activated PEG compound for crosslinking between sulfhydryl (—SH) groups in proteins and other thiol molecules. The maleimide groups at either end of the PEG3 spacer react specifically and efficiently with reduced sulfhydryls at pH 6.5-7.5 to form stable thioether bonds.
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. Thermo Scientific Pierce SM (PEG) n is a series of amine-to-sulfhydryl crosslinkers that differ in length from 17.6 to 95.2 angstroms as a result of polyethylene glycol spacer arms containing n equals 2 to 24 ethylene glycol units.
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.
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.
NHS-PEG4-Biotin is an example of an amine-reactive biotinylation reagent with a PEG spacer arm. Thermo Scientific EZ-Link NHS-PEG4-Biotin is a pegylated, water-soluble reagent for simple and efficient biotin labeling of antibodies, proteins and other primary amine-containing macromolecules.
Maleimide-PEG11-Biotin is an example of a sulfhydryl-reactive biotinylation reagent with a PEG spacer arm. Thermo Scientific EZ-Link Maleimide-PEG11-Biotin is a long, maleimide-activated, sulfhydryl-reactive biotinylation reagent that includes an 11-unit polyethylene glycol spacer arm for increased water-solubility and reach.
Hydrazide-PEG4-Biotin product is a carbohydrate-reactive biotinylation reagent with a PEG spacer arm. Thermo Scientific EZ-Link Hydrazide-PEG4-Biotin (also called Biotin-PEG4-Hydrazide) is an aldehyde-reactive biotinylation reagent that contains a 4-unit polyethylene glycol (PEG) spacer arm for increased hydrophilicity.
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. The example below illustrates the use of Invitrogen DyLight 680-4xPEG in an in vivo near-infrared fluorescence imaging experiment.
DyLight 680-4xPEG Dye exhibits rapid biodistribution and tissue clearance.
(A). Mice were injected with hydrolyzed DyLight 680-4xPEG Dye via the retro-orbital plexus, and NIR in vivo images were captured pre (Pre)- and post-injection at the indicated time points. Data indicate that the dye is rapidly distributed throughout the mouse body and signicantly cleared between 30m and 1h post-injection. (B) After the final time point, animals were sacrificed and tissues collected for ex vivo imaging to confirm dye accumulation in the kidneys and GI tract, indicative of clearance via the excretory system. Heat map legends indicate signal intensity.
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.
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. Thermo Scientific Pierce MS (PEG) n reagents are methyl-terminated, polyethylene glycol compounds (n equals 4 to 24 PEG units) activated as NHS esters for covalent pegylation of primary amines on proteins (e.g., lysines) or assay surfaces.
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.
MM (PEG) n compounds are linear 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 two PEG lengths (n = 12 and 24).
Sulfhydryl-reactive pegylation reagents. Thermo Scientific Pierce MM (PEG) 12 is a representative sulfhydryl-reactive pegylation reagent. It is a methyl-terminated, polyethylene glycol compound (12 PEG units) activated with a maleimide group for covalent pegylation of sulfhydryls on proteins (e.g., cysteines) or assay surfaces.
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.
Carboxyl-amine pegylation reagents. Thermo Scientific Pierce CA (PEG) 4 is a representative carboxyl-amine pegylation reagent. It is a pegylated amino acid of the form carboxy-PEG-amine, containing 4 polyethylene glycol units, and is useful for a variety of surface- and molecule-pegylation applications.
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For Research Use Only. Not for use in diagnostic procedures.