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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

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.

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

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

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

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.

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.

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

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.

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.

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)

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.

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.

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™ 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.