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GalNAz (N-azidoacetylgalactosamine tetraacylated) (Thermo Scientific™)

Thermo Scientific Pierce GalNAz (N-azidoacetylgalactosamine-tetraacylated) is an azide-labeled sugar that provides a highly specific approach for studying glycoproteins through in vivo metabolic labeling and chemoselective ligation.

Features of Azido-Sugars:

Bioorthogonal—the azido group is small, nonreactive and absent from living systems; as such the azido-sugar compounds do not interfere with endogenous cellular pathways and substitute for their naturally occurring analogs
Compatible—reaction chemistry with phosphine compounds occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents
Chemoselective—azide and phosphine groups do not react or interfere with components of biological samples but conjugate to one another with high efficiency
Versatile—azide tag can be targeted for detection, immobilization, conjugation or affinity purification depending on which phosphine-activated compound it is reacted with

These sugars are azide-derivatives of naturally occurring monosaccharides that cells use to glycosylate proteins using post-translational modification biochemical pathways. The azide functional group is small and nonreactive with endogenous molecules. When supplied to cells, these compounds become incorporated by glycosylation events to effectively "tag" glycoproteins with the azide group. The azide group then can be specifically targeted for detection or conjugation using alkyne-activated reagents ("click" chemistry) or phosphine-activated reagents (Staudinger ligation).

When used in combination with phosphine-activated fluorescent dyes, biotin reagents, and or other compounds, these azido-modified sugars facilitate the investigation of cellular pathways involving glycosylation.

There are several classes of glycoproteins grouped by the type of carbohydrate and amino acid linkage site. N-linked glycosylation is a modification of asparagine amines, whereas O-linked glycosylation occurs through the hydroxyl of serine and threonine residues. The azido-modified sugars are metabolic substitutes for endogenous amino sugars. ManNAz is converted by cells to an azido sialic acid derivative that is used for N-linked glycosylation of cell surface proteins. GlcNAz and GalNAz are predominantly used to label the O-linked glycosylation (O-GlcNAc and O-GalNAc).

Related Products
GlcNAz (N-azidoacetylglucosamine tetraacylated)
ManNAz (N-azidoacetylmannosamine tetraacylated)

DyLight™ 488-Phosphine (Thermo Scientific™)

Thermo Scientific DyLight Fluor Phosphine Reagents are phosphine-activated fluorescent dyes for specific labeling and detection of azide-tagged molecules, which enables use of fluorescence imaging in metabolic labeling strategies.

When used in combination with azide labeling strategies, phosphine-activated DyLight Fluors enable selective fluorescent labeling for detection of protein interactions and post-translational modifications using fluorescence imaging technologies. The phosphine group conjugates to azide groups by the Staudinger reaction mechanism. Azide groups can be introduced into proteins or other cellular targets through in vivo labeling with azide-tagged derivatives of naturally occurring metabolic building blocks. Because neither phosphines nor azides are present in biological systems, they comprise a chemoselective (mutually specific) ligation pair for labeling and conjugation.

General features of the phosphine-activated DyLight Fluors:

Soluble—easily dissolves in water-miscible solvents (e.g., DMSO) for subsequent dilution in aqueous reaction mixtures with cell lysates and other biological samples
Compatible—reaction chemistry occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents, and does not interfere with fluorescence applications
Chemoselective—the phosphine reactive group is specific in biological samples for bioorthogonal azide-tagged molecules, ensuring that fluorescent labeling is specific
High-performance fluorescence—DyLight 488, 550 and 650 are intense, highly stable fluorophores for green, orange and red fluorescent detection. (see DyLight Fluors)

Related Products
DyLight™ 550-Phosphine
DyLight™ 650-Phosphine

GlycanAssure™ AutoXpress Kit (Applied Biosystems™)

The GlycanAssure AutoXpress Kit is designed for use on the AutoMate Express System for GlycanAssure AutoXpress Kits, for hands-free automation of the preparation of labelled glycans from glycoprotein samples. The kit includes GlycanAssure AutoXpress cartridges that contain pre-filled reagents and plastic tubes and tips to perform hands-free glycan release and labelling of up to 13 samples in one run.

The GlycanAssure AutoXpress cartridge reagents are based on the established GlycanAssure chemistry for N-glycan rapid release, labeling, and cleanup that applies a novel method for N-glycan release in a complete form. The GlycanAssure APTS N-glycan sample prep method consists of rapid deglycosylation using PNGase-F enzyme followed by magnetic bead-based glycan purification, glycan labeling with ATPS dye, and excess dye removal. The GlycanAssure AutoXpress Kit combined with the AutoMate Express System offers a single, automated N-glycan sample prep workflow for both high throughput (CE) and characterization (UHPLC) biopharma applications.

Key features of the combined kit/system include:
• Fully automated sample prep for N-glycan analysis
• Rapid results with cartridge-based reagents for glycan release and labelling
• High quality glycan data with reduced analyst error
• Labeled glycans can be analyzed on LC or CE platforms
• Small system footprint and low-cost automation platform make it adaptable in both development and QC labs
• Consistent data for easy method development and transfer across the globe

GlycanAssure™ AutoXpress Kit with CE Module

The GlycanAssure AutoXpress Kit is designed for use on the AutoMate Express System for GlycanAssure AutoXpress Kits, for hands-free automation of the preparation of labelled glycans from glycoprotein samples. The kit includes GlycanAssure AutoXpress cartridges that contain pre-filled reagents and plastic tubes and tips to perform hands-free glycan release and labelling of up to 13 samples in one run. This version of the kit contains a CE module with additional chemistries needed for CE analysis.

The GlycanAssure AutoXpress cartridge reagents are based on the established GlycanAssure chemistry for N-glycan rapid release, labeling, and cleanup that applies a novel method for N-glycan release in a complete form. The GlycanAssure APTS N-glycan sample prep method consists of rapid deglycosylation using PNGase-F enzyme followed by magnetic bead-based glycan purification, glycan labeling with ATPS dye, and excess dye removal. The GlycanAssure AutoXpress Kit combined with the AutoMate Express System offers a single, automated N-glycan sample prep workflow for both high throughput (CE) and characterization (UHPLC) biopharma applications.

Key features of the combined kit/system include:
• Fully automated sample prep for N-glycan analysis
• Rapid results with cartridge-based reagents for glycan release and labelling
• High quality glycan data with reduced analyst error
• Labeled glycans can be analyzed on LC or CE platforms
• Small system footprint and low-cost automation platform make it adaptable in both development and QC labs
• Consistent data for easy method development and transfer across the globe

EZ-Link™ Psoralen-PEG3-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link Psoralen-PEG3-Biotin enables biotinylation of DNA or RNA via UV-light-activated intercalation of the psoralen group with thymine- and other pyrimidine-containing bases to form a covalent bonds.

Features of EZ-Link Psoralen-PEG3-Biotin:

Biomolecular labeling—biotinylate DNA or RNA (approximately 1 biotin per 10 to 20 bases) without interfering with hybridization
Photo-reactive—covalent attachment occurs in the presence of UV light (~350nm) for 10 to 30 minutes
Psoralen-activated—reacts through a cyclo-addition mechanism with the 5,6 double bond in thymine- and other pyrimidine-containing bases
Pegylated—spacer arm contains a hydrophilic, 3-unit, polyethylene glycol (PEG) group
Enhances solubility—pegylation imparts water solubility to the biotinylated molecule, helping to prevent aggregation of biotinylated antibodies stored in solution
Long reach —spacer arm (total length added to target) is 36.9 angstroms, minimizing steric hindrance for binding interactions with streptavidin

Psoralen-PEG3-Biotin is a photoactivatable reagent for biotinylation of DNA or RNA. Upon activation with UV-Light (~350nm), the psoralen group reacts with thymine- and other pyrimidine-containing bases to form a covalent bond. The hydrophilic polyethylene glycol (PEG) spacer arm imparts water solubility that is transferred to the biotinylated molecule, thus reducing aggregation of labeled proteins stored in solution. The PEG spacer arm also gives this reagent a long and flexible connection to minimize steric hindrance involved with binding to avidin molecules.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.

Applications:
• Selective labeling of cell surface proteins (Ref.1)
• Biotinylation of hyaluronan (Ref.2)
• Labeling of endotoxin for receptor binding studies (Ref.3)

GlycanAssure™ HyPerformance APTS Kit with CE Module (Applied Biosystems™)

The GlycanAssure HyPerformance APTS Kit is an N‑glycan rapid‑release, labeling, and cleanup kit that offers an N-glycan sample prep workflow for high-throughput glycan characterization applications within biopharma. Combined with the Applied Biosystems 3500xL Genetic Analyzer or Thermo Scientific Vanquish UHPLC system, the GlycanAssure HyPerformance APTS Kit offers an end-to-end N-glycan analysis solution, from clone selection to lot release. This version of the kit contains a CE module with additional chemistries needed for CE analysis.

Key features include:
• Easy N-glycan sample prep workflow for both LC & CE analysis platforms
• Superior data quality with unparalleled robustness
• Proprietary denaturant delivering complete deglycosylation of glycoproteins
• Preservation of sialylation offering accurate quantitation of sialylated glycans
• High-throughput end-to-end N-glycan analysis significantly reducing time to results

Workflow
Glycosylation is one of the key critical quality attributes of glycoprotein (mAb)-based biotherapeutics. Current glycan analysis methods consist of labor-intensive sample preparation that involve the use of toxic reagents, time-consuming vacuum centrifugation steps, and multiple pipetting steps.

The GlycanAssure HyPerformance APTS workflow overcomes these limitations. It delivers complete deglocosylation using a proprietary denaturant and superior preservation of sialylation, an important quality attribute of glycoproteins. Accurate quantitation of sialylated glycans is critical in biosimilar development. Most common sample prep methods cause de-sialylation of the glycans, leading to inaccurate quantitation. In addition, LC based separation techniques poorly quantitate highly sialylated glycans because they elute at the end of the LC profile. The GlycanAssure HyPerformance kit offers excellent preservation of sialylated glycans and allows analysis on a CE instrument where highly sialylated glycans migrate at the beginning of the electropherogram, enabling accurate quantitation.

The released, labelled glycans can be analyzed with either the Applied BioSystems 3500xL Genetic Analyzer for Protein Quality Analysis for high throughput applications or Thermo Scientific Vanquish Flex UHPLC System for lower throughput applications, for an end-to-end glycan analysis solution from Thermo Fisher Scientific.

Kit description
The GlycanAssure HyPerformance APTS Kit is part of the GlycanAssure Glycan Analysis and Quantitation System and contains all reagents and buffers needed to analyze and quantitate N-glycans from glycoprotein samples. APTS is a traditional dye used for glycan labeling and the labeled glycans can be analyzed using capillary electrophoresis (CE) or liquid chromatography (LC) systems. The GlycanAssure sample preparation workflow is a streamlined, automatable method for the processing and analysis of 96 glycoprotein samples in ~5 hrs.

The GlycanAssure HyPerformance APTS kit contains:
• GlycanAssure core reagents
• GlycanAssure beads
• GlycanAssure APTS labeling reagents
• GlycanAssure kit user guide

DyLight™ 550-Phosphine (Thermo Scientific™)

Thermo Scientific DyLight Fluor Phosphine Reagents are phosphine-activated fluorescent dyes for specific labeling and detection of azide-tagged molecules, which enables use of fluorescence imaging in metabolic labeling strategies.

When used in combination with azide labeling strategies, phosphine-activated DyLight Fluors enable selective fluorescent labeling for detection of protein interactions and post-translational modifications using fluorescence imaging technologies. The phosphine group conjugates to azide groups by the Staudinger reaction mechanism. Azide groups can be introduced into proteins or other cellular targets through in vivo labeling with azide-tagged derivatives of naturally occurring metabolic building blocks. Because neither phosphines nor azides are present in biological systems, they comprise a chemoselective (mutually specific) ligation pair for labeling and conjugation.

General features of the phosphine-activated DyLight Fluors:

Soluble—easily dissolves in water-miscible solvents (e.g., DMSO) for subsequent dilution in aqueous reaction mixtures with cell lysates and other biological samples
Compatible—reaction chemistry occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents, and does not interfere with fluorescence applications
Chemoselective—the phosphine reactive group is specific in biological samples for bioorthogonal azide-tagged molecules, ensuring that fluorescent labeling is specific
High-performance fluorescence—DyLight 488, 550 and 650 are intense, highly stable fluorophores for green, orange and red fluorescent detection. (see DyLight Fluors)

Related Products
DyLight™ 488-Phosphine
DyLight™ 650-Phosphine

Pierce™ Iodination Reagent (1,3,4,6-tetrachloro-3α,6α-diphenyl-glycoluril) (Thermo Scientific™)

Thermo Scientific Pierce Iodination Reagent is 1,3,4,6-tetrachloro-3a,6a-diphenyl-glycoluril, an oxidizing agent that efficiently activates iodine for spontaneous incorporation into protein tyrosyl groups.

Features of Iodination Reagent:

• Extremely stable and virtually insoluble in water (Fraker and Smith, 1978)
• Solubility in chloroform permits "plating" aliquots of the reagent on the walls of the glass or plastic iodination vessels; plated vessels can be prepared in advance and stored in a dessicator
• Allows rapid iodination of proteins in an aqueous reaction mixture containing radioactive Na125I
• Side reactions are negligible
• No reducing reagent is required, because reactions are terminated by simply decanting the reaction solution away from the plated IODO-GEN Iodination Reagent
• Specific activities of 1 x 105 cpm of 125I per microgram of protein and labeling efficiencies of 60% are easily achieved
• Can be used to iodinate phenolic groups on crosslinkers or other protein modification reagents either before, or after, a protein, peptide or cell surface is reacted with the modification reagent
• Small amounts of protein can be iodinated (Tolan, et al., 1980)
• More efficient than Chloramine-T or Lactoperoxidase (Salacinski, 1981)
• Iodinate phenolic groups on crosslinking reagents (see list of compatible reagents)

Unlike chloramine-T, Pierce Iodination Reagent can be coated on glassware to enable solid-phase oxidative activation and iodine-labeling. As a result, reducing agents are not necessary in the procedure.

Applications:
• Recommended for use with 125Iodine (Millar and Smith, 1983)
• Substitutes effectively for the highly toxic TlCl3 for nucleic acid iodination (Piatyszek, et al. 1988)
• Oxidatively remove sulfhydryl groups from solution prior to protein assay (McClard, 1981)

Iodination involves the introduction of radioactive iodine (I-125 or I-131) into certain amino acids (usually tyrosines) in proteins and peptides. Iodination takes place at the positions ortho to the hydroxyl group on tyrosine; mono- or di-substitution can occur. When iodinatable sites such as tyrosines are absent or of limited accessibility in a protein, iodinatable phenolic sites can be introduced by using the Bolton-Hunter Reagents (SHPP and Sulfo-SHPP). Certain crosslinkers also contain iodinatable tyrosyl groups in their spacer arms.

Radioactive 125-I or 131-I can be incorporated into proteins either by enzymatic or chemical oxidation. In the chemical oxidation method, sodium iodide is converted to its corresponding reactive iodine form, which then spontaneously incorporates into tyrosyl groups. While necessary for iodine activation, oxidizing reagents are potentially damaging to proteins. Pierce Iodination Reagent (previously known as IODO-GEN Reagent) is milder than chloramine-T, yet equally effective for iodine activation. Using the reagent in a solid-phase procedure makes iodination even more gentle on proteins.

Sulfo-SHPP (Water-Soluble Bolton-Hunter Reagent) (Thermo Scientific™)

Thermo Scientific Pierce Sulfo-SHPP, sulfosuccinimidyl-3 -(4-hydroxypheynyl) propionate, also known as water-soluble Bolton-Hunter Reagent, conjugates tyrosine-like functional groups to primary amines to increase the number of tyrosyl groups that can be iodinated by iodine-125 labeling procedures.

Features of Sulfo-SHPP:

• Iodinate before or after coupling Sulfo-SHPP to the molecule of interest
• Useful for introducing tyrosyl groups on proteins sensitive to small amounts of organic solvent
• Useful for labelling cell surfaces without exposing the cells to membrane permeable solvents

Alexa Fluor™ 488 Azide (Alexa Fluor™ 488 5-Carboxamido-(6-Azidohexanyl), Bis(Triethylammonium Salt)), 5-isomer (Invitrogen™)

The green-fluorescent Alexa Fluor® 488 azide is reactive with terminal alkynes via a copper-catalyzed click reaction. In addition to being an exceedingly bright and photostable fluorophore for use with flow cytometry, microscopy and HCS, Alexa Fluor® 488 can also be utilized as a bio-orthogonal or biologically unique hapten for use in applications requiring signal amplification.

Oregon Green™ 488 Azide (Oregon Green™ 6-Carboxamido-(6-Azidohexanyl), Triethylammonium Salt), 6-isomer (Invitrogen™)

The green-fluorescent Oregon Greenr® 488 azide is reactive with terminal alkynes via a copper-catalyzed click reaction. In addition to being a bright and photostable alternative to fluorescein for use in flow cytometry, microscopy and HCS, Oregon Green® 488 can also be utilized as a bio-orthogonal or biologically unique hapten for use in applications requiring signal amplification.

ManNAz (N-azidoacetylmannosamine tetraacylated) (Thermo Scientific™)

Thermo Scientific Pierce ManNAz (N-azidoacetylmannosamine-tetraacylated) is an azide-labeled sugar that provides a highly specific approach for studying glycoproteins through in vivo metabolic labeling and chemoselective ligation.

Features of Azido-Sugars:

Bioorthogonal—the azido group is small, nonreactive and absent from living systems; as such the azido-sugar compounds do not interfere with endogenous cellular pathways and substitute for their naturally occurring analogs
Compatible—reaction chemistry with phosphine compounds occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents
Chemoselective—azide and phosphine groups do not react or interfere with components of biological samples but conjugate to one another with high efficiency
Versatile—azide tag can be targeted for detection, immobilization, conjugation or affinity purification depending on which phosphine-activated compound it is reacted with

These sugars are azide-derivatives of naturally occurring monosaccharides that cells use to glycosylate proteins using post-translational modification biochemical pathways. The azide functional group is small and nonreactive with endogenous molecules. When supplied to cells, these compounds become incorporated by glycosylation events to effectively "tag" glycoproteins with the azide group. The azide group then can be specifically targeted for detection or conjugation using alkyne-activated reagents ("click" chemistry) or phosphine-activated reagents (Staudinger ligation).

When used in combination with phosphine-activated fluorescent dyes, biotin reagents, and or other compounds, these azido-modified sugars facilitate the investigation of cellular pathways involving glycosylation.

There are several classes of glycoproteins grouped by the type of carbohydrate and amino acid linkage site. N-linked glycosylation is a modification of asparagine amines, whereas O-linked glycosylation occurs through the hydroxyl of serine and threonine residues. The azido-modified sugars are metabolic substitutes for endogenous amino sugars. ManNAz is converted by cells to an azido sialic acid derivative that is used for N-linked glycosylation of cell surface proteins. GlcNAz and GalNAz are predominantly used to label the O-linked glycosylation (O-GlcNAc and O-GalNAc).

Related Products
GlcNAz (N-azidoacetylglucosamine tetraacylated)
GalNAz (N-azidoacetylgalactosamine tetraacylated)

DyLight™ 650-Phosphine (Thermo Scientific™)

Thermo Scientific DyLight Fluor Phosphine Reagents are phosphine-activated fluorescent dyes for specific labeling and detection of azide-tagged molecules, which enables use of fluorescence imaging in metabolic labeling strategies.

When used in combination with azide labeling strategies, phosphine-activated DyLight Fluors enable selective fluorescent labeling for detection of protein interactions and post-translational modifications using fluorescence imaging technologies. The phosphine group conjugates to azide groups by the Staudinger reaction mechanism. Azide groups can be introduced into proteins or other cellular targets through in vivo labeling with azide-tagged derivatives of naturally occurring metabolic building blocks. Because neither phosphines nor azides are present in biological systems, they comprise a chemoselective (mutually specific) ligation pair for labeling and conjugation.

General features of the phosphine-activated DyLight Fluors:

Soluble—easily dissolves in water-miscible solvents (e.g., DMSO) for subsequent dilution in aqueous reaction mixtures with cell lysates and other biological samples
Compatible—reaction chemistry occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents, and does not interfere with fluorescence applications
Chemoselective—the phosphine reactive group is specific in biological samples for bioorthogonal azide-tagged molecules, ensuring that fluorescent labeling is specific
High-performance fluorescence—DyLight 488, 550 and 650 are intense, highly stable fluorophores for green, orange and red fluorescent detection. (see DyLight Fluors)

Related Products
DyLight™ 488-Phosphine
DyLight™ 550-Phosphine

Alexa Fluor™ 647 Azide, Triethylammonium Salt (Invitrogen™)

The far red-fluorescent Alexa Fluor® 647 azide is reactive with terminal alkynes via a copper-catalyzed click reaction. The bright and photostable fluorophore can be for used with flow cytometry, microscopy and HCS

Pierce™ Iodination Tubes (Thermo Scientific™)

Thermo Scientific Pierce Iodination Tubes are 12 x 75mm glass test tubes that are pre-coated at the bottom with Pierce Iodination Reagent, which activates iodine-125 for protein or peptide iodination procedures.

Features of Iodination Tubes:

• Eliminates the tedious reagent surface coating step
• Consistent, flake-resistant coating of the iodination reagent
• Generates more than sufficient oxidative capacity for a typical protocol
• Allows iodinations to be carried out directly in the tube or in two-steps (see next)

The pre-coated iodination tubes provide for convenient and efficient iodine labeling without the hassles and damaging oxidative effects normally associated with chloramine T and other solution-based methods. Coating glass surfaces to support iodine labeling experiments is difficult to reliably accomplish. These pre-coated glass tubes eliminate that difficult preparative step to ensure that iodination experiments are as simple as possible.

Iodination involves the introduction of radioactive iodine into certain amino acids (usually tyrosines) in proteins and peptides. Iodination takes place at the positions ortho to the hydroxyl group on tyrosine; mono- or di-substitution can occur. When iodinatable sites such as tyrosines are absent or of limited accessibility in a protein, iodinatable phenolic sites can be introduced by using the Bolton-Hunter Reagents (SHPP and Sulfo-SHPP). Certain crosslinkers also contain iodinatable tyrosyl groups in their spacer arms.

Radioactive 125-I or 131-I can be incorporated into proteins either by enzymatic or chemical oxidation. In the chemical oxidation method, sodium iodide is converted to its corresponding reactive iodine form, which then spontaneously incorporates into tyrosyl groups. While necessary for iodine activation, oxidizing reagents are potentially damaging to proteins. Pierce Iodination Reagent (previously known as IODO-GEN Reagent) is milder than chloramine-T, yet equally effective for iodine activation. The solid-phase arrangement provided by the pre-coated tubes further optimizes iodination and minimizes damaging effects.

Benefits of two-step labeling method (Chizzonite, 1996):
• Eliminates oxidative damage to proteins by avoiding direct contact with iodination reagent
• No losses from nonspecific protein binding to the coated tube surface
• Flexibility to conduct iodination in a wide variety of vessels
• Easy scale-up or scale-down of the iodination protocol
• Gentle iodine-labeling of a wide range of proteins, peptides or cell surfaces
• Compatibility with common detergents

Alexa Fluor™ 488 Alkyne (Alexa Fluor™ 488 5-Carboxamido-(Propargyl), Bis(Triethylammonium Salt)), 5-isomer (Invitrogen™)

The green-fluorescent Alexa Fluor® 488 alkyne is reactive with azides via a copper-catalyzed click reaction. In addition to being an exceedingly bright and photostable fluorophore for use with flow cytometry, microscopy and HCS, Alexa Fluor® 488 can also be utilized as a bio-orthogonal or biologically unique hapten for use in applications requiring signal amplification.

GlycanAssure™ APTS Kit (Applied Biosystems™)

The GlycanAssure™ APTS Kit is part of the GlycanAssure™ Glycan Analysis and Quantitation System and contains all reagents and buffers needed to analyze and quantitate N-glycans from glycoprotein samples. The GlycanAssure APTS N-glycan sample prep method consists of rapid deglycosylation using PNGase-F enzyme followed by magnetic bead-based glycan purification, glycan labeling with ATPS dye, and excess dye removal. APTS is a traditional dye used for glycan labeling for the past several years, and the labeled glycans can be analyzed using capillary electrophoresis (CE) or liquid chromatography (LC) system. The GlycanAssure sample prep workflow does not include time-consuming vacuum centrifugation steps or the use of toxic sodium cyanoborohydride like more traditional workflows, resulting in a streamlined, automatable method for the processing and analysis of 96 samples in ~9 hrs (< 4 hrs hands-on time).

The GlycanAssure APTS kit contains:
• GlycanAssure core reagents
• GlycanAssure beads
• GlycanAssure APTS labeling reagents
• GlycanAssure kits user guide

GlycanAssure Glycan Analysis and Quantitation System
The GlycanAssure Glycan Analysis and Quantitation System is the first glycan analysis system that provides both high throughput and high data quality through an integrated glycan analysis platform that helps save labor, time, and cost of analysis. The GlycanAssure system offers simple and easy magnetic bead-based sample preparation with multiple fluorescent dyes for glycan labeling, multi-capillary Sanger sequencing instruments for high-throughput CE-LIF-based glycan analysis, and assay-specific software for fast data analysis and reporting.

As part of the GlycanAssure Glycan Analysis and Quantitation System, the 3500 Genetic Analyzer for Protein Quality Analysis and 3500xL Genetic Analyzer for Protein Quality Analysis are multi-capillary CE instruments that enable parallel analysis of 8 or 24 samples on a 50-cm capillary array for high-throughput and high-resolution glycan analysis. The ability to analyze samples in parallel avoids the need for shortened run times to achieve high throughput. The 3500 Series systems for protein quality analysis come with integrated assay-specific GlycanAssure data acquisition and data analysis software for optimum performance and user experience.

GlycanAssure™ HyPerformance APTS Kit (Applied Biosystems™)

The GlycanAssure HyPerformance APTS Kit is an N‑glycan rapid‑release, labeling, and cleanup kit that offers an N-glycan sample prep workflow for high-throughput glycan characterization applications within biopharma. Combined with the Applied Biosystems 3500xL Genetic Analyzer or Thermo Scientific Vanquish UHPLC system, the GlycanAssure HyPerformance APTS Kit offers an end-to-end N-glycan analysis solution, from clone selection to lot release.

Key features include:
• Easy N-glycan sample prep workflow for both LC & CE analysis platforms
• Superior data quality with unparalleled robustness
• Proprietary denaturant delivering complete deglycosylation of glycoproteins
• Preservation of sialylation offering accurate quantitation of sialylated glycans
• High-throughput end-to-end N-glycan analysis significantly reducing time to results

Workflow
Glycosylation is one of the key critical quality attributes of glycoprotein (mAb)-based biotherapeutics. Current glycan analysis methods consist of labor-intensive sample preparation that involve the use of toxic reagents, time-consuming vacuum centrifugation steps, and multiple pipetting steps.

The GlycanAssure HyPerformance APTS workflow overcomes these limitations. It delivers complete deglocosylation using a proprietary denaturant and superior preservation of sialylation, an important quality attribute of glycoproteins. Accurate quantitation of sialylated glycans is critical in biosimilar development. Most common sample prep methods cause de-sialylation of the glycans, leading to inaccurate quantitation. In addition, LC based separation techniques poorly quantitate highly sialylated glycans because they elute at the end of the LC profile. The GlycanAssure HyPerformance kit offers excellent preservation of sialylated glycans and allows analysis on a CE instrument where highly sialylated glycans migrate at the beginning of the electropherogram, enabling accurate quantitation.

The released, labelled glycans can be analyzed with either the Applied BioSystems 3500xL Genetic Analyzer for Protein Quality Analysis for high throughput applications or Thermo Scientific Vanquish Flex UHPLC System for lower throughput applications, for an end-to-end glycan analysis solution from Thermo Fisher Scientific.

Kit description
The GlycanAssure HyPerformance APTS Kit is part of the GlycanAssure Glycan Analysis and Quantitation System and contains all reagents and buffers needed to analyze and quantitate N-glycans from glycoprotein samples. APTS is a traditional dye used for glycan labeling and the labeled glycans can be analyzed using capillary electrophoresis (CE) or liquid chromatography (LC) systems. The GlycanAssure sample preparation workflow is a streamlined, automatable method for the processing and analysis of 96 glycoprotein samples in ~5 hrs.

The GlycanAssure HyPerformance APTS kit contains:
• GlycanAssure core reagents
• GlycanAssure beads
• GlycanAssure APTS labeling reagents
• GlycanAssure kit user guide

Tetramethylrhodamine (TAMRA) Alkyne (5-Carboxytetramethylrhodamine, Propargylamide), 5-isomer (Invitrogen™)

The red-fluorescent tetramethylrhodamine (TAMRA) alkyne can be reacted with azides via a copper-catalyzed click reaction. Click chemistry describes a class of chemical reactions that use bio-orthogonal or biologically unique moities to label and detect a molecule of interest using a two-step procedure. The two-step reaction procedure involves a copper-catalyzed triazole formation of an azide and an alkyne. Click reactions have several characteristics: the reaction between the detection moieties is efficient; no extreme temperatures or solvents are required; the reaction product is stable; the components of the reaction are bioinert; and perhaps most importantly, no side reactions occur – the label and detection tags react selectively and specifically with one another. Unlike traditional chemical reactions utilizing succinimidyl esters or maleimides that target amines and sulfhydryls – functional groups that are not unique – click chemistry-labeled molecules can be applied to complex biological samples and be detected with unprecedented sensitivity due to extremely low background.

EZ-Link™ Phosphine-PEG3-Biotin (Thermo Scientific™)

Thermo Scientific Pierce EZ-Link Phosphine-PEG3-Biotin is a biotinylation reagent for labeling azide-containing molecules, which enables biotin-based detection and affinity purification of molecules via Staudinger ligation strategies.

Features of EZ-Link Phosphine-PEG3-Biotin:

Soluble—easily dissolves in water-miscible solvents (e.g., DMSO) for subsequent dilution in aqueous reaction mixtures with cell lysates and other biological samples
Compatible—reaction chemistry occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents
Chemoselective—the phosphine reactive group is specific in biological samples for bioorthogonal azide-tagged molecules, ensuring that biotinylation is specific
PEG spacer—polyethylene glycol spacer arm helps maintain solubility of labeled molecules and decreases steric hindrance for affinity-binding to avidin, streptavidin or NeutrAvidin Protein

When used in combination with azide labeling strategies, this compound enables detection or affinity purification of protein interactions and post-translational modifications using streptavidin probes or streptavidin agarose resins. The phosphine group of Phosphine-PEG3-Biotin conjugates to azide groups by the Staudinger reaction mechanism. Azide groups can be introduced into proteins or other cellular targets through in vivo labeling with azide-tagged derivatives of naturally occurring metabolic building blocks. Because neither phosphines nor azides are present in biological systems, they comprise a chemoselective (mutually specific) ligation pair for labeling and conjugation.

GlcNAz (N-azidoacetylglucosamine tetraacylated) (Thermo Scientific™)

Thermo Scientific Pierce GlcNAz (N-azidoacetylglucosamine-tetraacylated) is an azide-labeled sugar that provides a highly specific approach for studying glycoproteins through in vivo metabolic labeling and chemoselective ligation.

Features of Azido-Sugars:

Bioorthogonal—the azido group is small, nonreactive and absent from living systems; as such the azido-sugar compounds do not interfere with endogenous cellular pathways and substitute for their naturally occurring analogs
Compatible—reaction chemistry with phosphine compounds occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents
Chemoselective—azide and phosphine groups do not react or interfere with components of biological samples but conjugate to one another with high efficiency
Versatile—azide tag can be targeted for detection, immobilization, conjugation or affinity purification depending on which phosphine-activated compound it is reacted with

These sugars are azide-derivatives of naturally occurring monosaccharides that cells use to glycosylate proteins using post-translational modification biochemical pathways. The azide functional group is small and nonreactive with endogenous molecules. When supplied to cells, these compounds become incorporated by glycosylation events to effectively "tag" glycoproteins with the azide group. The azide group then can be specifically targeted for detection or conjugation using alkyne-activated reagents ("click" chemistry) or phosphine-activated reagents (Staudinger ligation).

When used in combination with phosphine-activated fluorescent dyes, biotin reagents, and or other compounds, these azido-modified sugars facilitate the investigation of cellular pathways involving glycosylation.

There are several classes of glycoproteins grouped by the type of carbohydrate and amino acid linkage site. N-linked glycosylation is a modification of asparagine amines, whereas O-linked glycosylation occurs through the hydroxyl of serine and threonine residues. The azido-modified sugars are metabolic substitutes for endogenous amino sugars. ManNAz is converted by cells to an azido sialic acid derivative that is used for N-linked glycosylation of cell surface proteins. GlcNAz and GalNAz are predominantly used to label the O-linked glycosylation (O-GlcNAc and O-GalNAc).

Related Products
ManNAz (N-azidoacetylmannosamine tetraacylated)
GalNAz (N-azidoacetylgalactosamine tetraacylated)

Alexa Fluor™ 594 Alkyne (Alexa Fluor™ 594 Carboxamido-(5-(and 6-)Propargyl), Bis(Triethylammonium Salt)), mixed isomers (Invitrogen™)

The red-fluorescent Alexa Fluor® 594 alkyne can be reacted with azides via a copper-catalyzed click reaction. Click chemistry describes a class of chemical reactions that use bio-orthogonal or biologically unique moities to label and detect a molecule of interest using a two-step procedure. The two-step reaction procedure involves a copper-catalyzed triazole formation of an azide and an alkyne. Click reactions have several characteristics: the reaction between the detection moieties is efficient; no extreme temperatures or solvents are required; the reaction product is stable; the components of the reaction are bioinert; and perhaps most importantly, no side reactions occur – the label and detection tags react selectively and specifically with one another. Unlike traditional chemical reactions utilizing succinimidyl esters or maleimides that target amines and sulfhydryls – functional groups that are not unique – click chemistry-labeled molecules can be applied to complex biological samples and be detected with unprecedented sensitivity due to extremely low background.

Alexa Fluor™ 647 Alkyne, Triethylammonium Salt (Invitrogen™)

The far red-fluorescent Alexa Fluor® 647 alkyne is reactive with azides via a copper-catalyzed click reaction. The bright and photostable fluorophore can be for used with flow cytometry, microscopy and HCS

Click-iT™ Protein Enrichment Kit, for click chemistry capture of azide-modified proteins (Invitrogen™)

The Click-iT® Protein Enrichment Kit for the efficient capture of click chemistry based azide-modified proteins on a resin of alkyne agarose. Superior to biotin or lectin based enrichment approaches. Ideal for proteomics, biomarker discovery, posttranslational modification (PTM) analysis, and more. The azide modification can occur via metabolic feeding, enzymatic addition, or chemical modification. Click-Azide modified proteins, or their post-translationally modified forms, are enriched from complex protein extracts on the alkyne-agarose resin supplied. Once anchored to the resin via copper catalyzed click chemistry, extensive washing yields a highly enriched population of nascent molecules that can be furthered characterized by mass spectrometry. The alkyne-agarose resin improves upon existing biotin approaches by>8 fold, with signal to noise of biotin = 3 while the Click-iT resin = 25.

Perfect upstream MS-based sample preparation technique for looking at changes in global protein expression and biomarker analysis.

The new Click-iT® enrichment resin affords many distinct advantages including::

  1. global differential profiling of multiple subclasses of posttranslationally modified (PTM) proteins and newly synthesized proteins


  2. improved signal to noise by eliminating nonspecific binding and increasing selectivity, thus improving detection of low abundant proteins


  3. accelerates PTM site identification leading to more rapid initiation of functional studies

  4. fully compatible with widely used MS techniques including iTRAC and ICAT.


Seamless integration of cell biology with protein chemistry.

Affordable, easy, no special equipment required.

To know what’s new in your cells use Click-iT®

Click-iT®: One Reaction, Endless Possibilities.

Tetramethylrhodamine (TAMRA) Azide (Tetramethylrhodamine 5-Carboxamido-(6-Azidohexanyl)), 5-isomer (Invitrogen™)

The red-fluorescent tetramethylrhodamine (TAMRA) azide can be reacted with terminal alkynes via a copper-catalyzed click reaction. Click chemistry describes a class of chemical reactions that use bio-orthogonal or biologically unique moities to label and detect a molecule of interest using a two-step procedure. The two-step reaction procedure involves a copper-catalyzed triazole formation of an azide and an alkyne. Click reactions have several characteristics: the reaction between the detection moieties is efficient; no extreme temperatures or solvents are required; the reaction product is stable; the components of the reaction are bioinert; and perhaps most importantly, no side reactions occur – the label and detection tags react selectively and specifically with one another. Unlike traditional chemical reactions utilizing succinimidyl esters or maleimides that target amines and sulfhydryls – functional groups that are not unique – click chemistry-labeled molecules can be applied to complex biological samples and be detected with unprecedented sensitivity due to extremely low background.

Pierce™ Iodination Beads (Thermo Scientific™)

Thermo Scientific Pierce Iodination Beads are 3mm-diameter polystyrene beads that are coated with an oxidizing reagent, which provides efficient activation of iodine-125 for protein or peptide iodination procedures.

Features of Iodination Beads:

• Derivatized, uniform, nonporous polystyrene beads
• Remarkably reproducible iodinations
• Iodine-125 incorporation as high as 99%; labeled protein recovery > 90%
• Functions over a broad pH and temperature range
• Iodinates in the presence of azides, detergents, urea and high salt
• Allows efficient iodination of cell membrane surface proteins
• More gentle method for iodination than soluble chloramine-T because there is no contact between the protein and the immobilized oxidizing agent (Markwell, 1982)
• Fast and easy to use…iodinations complete in 2-15 minutes
• Reaction stopped by simply removing beads from reaction mixture with tweezers or pasteur pipet; no reducing agent necessary to terminate reaction
• More control over the incubation time
• Iodinate up to 500 µg of tyrosine-containing peptide or protein/bead
• Can be used to quantitatively iodinate histidine at pH 8.22

The coated iodination beads provide for convenient and efficient iodine-labeling without the hassles and damaging oxidative effects normally associated with chloramine T and other solution-based methods. One or two beads can be added per milliliter of protein solution and then easily removed following the labeling reaction, thereby completely removing the oxidizing reagent from the protein.

Iodination involves the introduction of radioactive iodine into certain amino acids (usually tyrosines) in proteins and peptides. Iodination takes place at the positions ortho to the hydroxyl group on tyrosine; mono- or di-substitution can occur. When iodinatable sites such as tyrosines are absent or of limited accessibility in a protein, iodinatable phenolic sites can be introduced by using the Bolton-Hunter Reagents (SHPP and Sulfo-SHPP). Certain crosslinkers also contain iodinatable tyrosyl groups in their spacer arms.

Radioactive 125-I or 131-I can be incorporated into proteins either by enzymatic or chemical oxidation. In the chemical oxidation method, sodium iodide is converted to its corresponding reactive iodine form, which then spontaneously incorporates into tyrosyl groups. While necessary for iodine activation, oxidizing reagents are potentially damaging to proteins. The solid-phase arrangement provided by the Iodination Beads (previously called IODO-BEADS) minimizes damaging effects.

EZ-Link™ TFPA-PEG3-Biotin (Thermo Scientific™)

Thermo Scientific EZ-Link TFPA-PEG3-Biotin is an efficient, photoactivatable reagent based on tetrafluorophenyl azide for biotinylation and includes a 3-unit polyethylene glycol (PEG) spacer arm.

Features of EZ-Link TFPA-PEG3-Biotin:

Biomolecular labeling—biotinylate proteins, DNA, RNA and many other macromolecules, even if they do not possess primary amines or sulfhydryl groups
Photo-reactive—perfluorophenyl azido group activates upon exposure to ultraviolet light to form covalent bonds with nucleophiles and many other chemical groups
Pegylated—spacer arm contains a hydrophilic, 3-unit, polyethylene glycol (PEG) group
Enhances solubility—pegylation imparts water solubility to the biotinylated molecule, helping to prevent aggregation of biotinylated antibodies stored in solution
Irreversible—forms permanent thioether bonds; spacer arm cannot be cleaved
Solubility—best to dissolve in DMSO or DMF before further dilution in aqueous buffers
Long reach —spacer arm (total length added to target) is 33.4 angstroms, minimizing steric hindrance for binding interactions with streptavidin

TFPA-PEG3-Biotin is a photoactivatable reagent for biotinylation of antibodies, proteins and many other kinds of macromolecules. The tetrafluorophenyl azide (TFPA) group activates upon exposure to UV-Light (maximum absorptivity is at 320 nm) to insert covalently at sites containing C-H or N-H bonds. The hydrophilic polyethylene glycol (PEG) spacer arm imparts water solubility that is transferred to the biotinylated molecule, thus reducing aggregation of labeled molecules stored in solution. The PEG spacer arm also gives this reagent a long and flexible connection to minimize steric hindrance involved with binding to avidin molecules.

We manufacture biotin reagents to ensure the highest possible overall product integrity, consistency, and performance for the intended research applications.

Biotinylation reagents differ in reactivity, length, solubility, cell permeability and cleavability. Several different types of photoreactive compounds are available. Aryl azide reagents activate upon exposure to ultraviolet light initiate addition reactions with double bonds, insertion into C–H and N–H sites, or subsequent ring expansion to react with a nucleophile (e.g., primary amines).

Alexa Fluor™ 594 Azide (Alexa Fluor™ 594 Carboxamido-(6-Azidohexanyl), Triethylammonium Salt), mixed isomers (Invitrogen™)

The red-fluorescent Alexa Fluor® 594 azide can be reacted with terminal alkynes via a copper-catalyzed click reaction. Click chemistry describes a class of chemical reactions that use bio-orthogonal or biologically unique moities to label and detect a molecule of interest using a two-step procedure. The two-step reaction procedure involves a copper-catalyzed triazole formation of an azide and an alkyne. Click reactions have several characteristics: the reaction between the detection moieties is efficient; no extreme temperatures or solvents are required; the reaction product is stable; the components of the reaction are bioinert; and perhaps most importantly, no side reactions occur – the label and detection tags react selectively and specifically with one another. Unlike traditional chemical reactions utilizing succinimidyl esters or maleimides that target amines and sulfhydryls – functional groups that are not unique – click chemistry-labeled molecules can be applied to complex biological samples and be detected with unprecedented sensitivity due to extremely low background.

Alexa Fluor™ 555 alkyne, Triethylammonium Salt (Invitrogen™)

The orange-fluorescent Alexa Fluor® 555 alkyne is designed to react with azide-containing molecules via the fast, selective and extremely efficient copper-catalyzed “click" reaction.

Alexa Fluor™ 555 Azide, Triethylammonium Salt (Invitrogen™)

The orange-fluorescent Alexa Fluor® 555 azide is designed to react with alkyne-containing molecules via the fast, selective and extremely efficient copper-catalyzed “click" reaction.