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EZ-Link™ NHS-PEG4-Biotin, No-Weigh™ Format (Thermo Scientific™)

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.

Features of EZ-Link NHS-PEG4-Biotin:

Protein labeling—biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins
Amine-reactive—reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the amino-termini of polypeptides
Pegylated – spacer arm contains a hydrophilic, 4-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 amide bonds; spacer arm cannot be cleaved
Long reach – spacer arm (total length added to target) is 29 angstroms; this reduces steric hindrance when binding to avidin molecules

NHS-PEG4-Biotin is a long (29.0Å), pegylated, water-soluble, NHS-ester biotinylation reagent to label amines and maximize solubility of antibodies and other proteins. The N-hydroxysuccinimide ester (NHS) group reacts specifically and efficiently with lysine and N-terminal amino groups to form stable amide bonds. 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 the reagent a long and flexible connection to minimize steric hindrance for binding to avidin molecules.

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

N-Hydroxysulfosuccinimide (NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amino groups (-NH2) in alkaline buffers to form stable amide bonds. Proteins (e.g., antibodies) typically have several primary amines that are available as targets for labeling, including the side chain of lysine (K) residues and the N-terminus of each polypeptide.

Varieties of biotin NHS-ester reagents differ in length, solubility, cell permeability and cleavability. Non-sulfonated NHS-biotins are cell permeable but must be dissolved in organic solvent such as DMSO or DMF. Sulfo-NHS biotins (and those with pegylated spacers) are directly water soluble but not membrane permeable. Varieties containing disulfide bonds can be cleaved using reducing agents, enabling the biotin group to be disconnected from the labeled protein.

Related Products
EZ-Link™ NHS-PEG4 Biotinylation Kit
EZ-Link™ Micro NHS-PEG4-Biotinylation Kit

DMS (dimethyl suberimidate) (Thermo Scientific™)

Thermo Scientific Pierce DMS is dimethyl suberimidate, a membrane-permeable crosslinker that contains an amine-reactive imidoester group at each end of an 8-atom spacer arm.

Features of dimethyl suberimidate:

Reactive groups: imidoester (both ends)
Reactive towards: amino groups (primary amines)
• Longest of three similar imidoester crosslinkers, which include DMA, DMP and DMS
• Water-soluble
• Imidoesters react with amines at alkaline pH values (pH 8-10) to form amidine bond
• Amidine bond retains net charge character of protein primary amine to which it reacts
• Reversible at high pH values
• Tool for study of quarternary structure of proteins

Product References
Crosslinker Application Guide -- search for recent literature references for this product

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DMA (dimethyl adipimidate)
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EMCH (N-ε-maleimidocaproic acid hydrazide) (Thermo Scientific™)

Thermo Scientific Pierce EMCH is a mid-length, maleimide-and-hydrazide crosslinker for conjugating sulfhydryls (cysteines) to carbonyls (aldehyde or ketones, such as those formed by oxidation of glycoprotein carbohydrates).

Features of EMCH:

Reactive groups: maleimide and hydrazide
Reactive towards: sulfhydryl groups and carbonyl (aldehyde) groups
• Mid-length (11.8Å), sulfhydryl-to-aldehyde crosslinker with simple spacer arm (noncleavable)
• Maleimide group reacts with sulfhydryl groups to form stable thioether linkages
• Hydrazide group conjugates to oxidized sugars of glycoproteins and carbohydrates
• Use sodium meta-periodate to oxidize glycosylation (e.g., sialic acid) to reactive aldehyde groups
• Use with EDC to conjugate primary amine of hydrazide group to carboxyl groups

3,3'-N-[ε-Maleimidocaproic acid] hydrazide, trifluoroacetic acid salt (EMCH) is a water-soluble, heterobifunctional, membrane permeable crosslinker. It contains a sulfhydryl reactive maleimide group and carbonyl reactive hydrazide group at each end of a 6-carbon spacer arm. Maleimides react with sulfhydryls at pH 6.5-7.5 to form stable thiol ether bonds, along with release of the maleimide leaving group. Proteins with cysteine residues not involved in disulfide bond formation are targets for maleimide reactive groups. Carbonyl groups, whilst not found naturally on proteins can be formed by ring sugar reduction to form aldehydes which react with hydrazides at pH5.5-7.5.

Applications:
• Chemical crosslinking of glycoproteins to sulfhydryl-containing molecules
• Use with EDC for reactivity toward carboxyl groups

Specifications of EMCH:
We manufacture EMCH to the highest specifications to produce the most specific bioconjugates, ensure the integrity of your data and to provide you with the highest degree of consistency. Each lot of EMCH is tested to meet the following minimum specifications:

• NMR purity: >90%
• Melting point: 99-110°C
• Identity: IR scan shows only peaks characteristic of the structure and functional groups of EMCH

Product References:
Crosslinker Application Guide -- search for recent literature references for this product

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L-Photo-Leucine (Thermo Scientific™)

Thermo Scientific Pierce L-Photo-Leucine is an analog of L-leucine that can be incorporated into proteins during synthesis and then light-activated to covalently crosslink interacting proteins in cells.

Features of L-Photo-Leucine:

In vivo labeling—incorporate photo-reactive group into proteins using normal cellular machinery of live cells
In vivo crosslinking—find interacting proteins in the native cellular environment
Increased specificity—crosslink interacting proteins correctly positioned at their interfaces within protein interaction domains
Efficient recovery—90% protein recovery in cell lysates after crosslinking
Compatible—crosslink proteins expressed in a wide variety of cell lines, including HeLa, 293T, COS7, U2OS, A549, A431, HepG2, NIH 3T3 and C6
Easy to use—reagents are photo-stable under normal laboratory lighting, eliminating the need to work in the dark

Thermo Scientific L-Photo-Leucine is an analog of L-leucine that can be endogenously incorporated into the primary sequence of proteins during synthesis and then ultraviolet light (UV)-activated to covalently crosslink proteins within protein-protein interaction domains in their native environment within live cells. The powerful method enables both stable and transient protein interactions in cells to be studied and characterized without the use of completely foreign chemical crosslinkers and associated reagent solvents during the interaction experiment that can adversely aspects of the cell biology being studied.

When used in combination with specially formulated limiting media that is devoid of leucine, the photo-activatable derivative is treated like a naturally occurring amino acid by the protein synthesis machinery within the cell. As a result, it can be substituted for leucine in the primary sequence of proteins during catabolism and growth. Photo-leucine derivatives contain diazirine rings that activate when exposed to UV light to become reactive intermediates that form covalent bonds with nearby protein side chains and backbones. Naturally associating binding partners within the cell can be instantly trapped by photoactivation of the diazirine-containing proteins in the cultured cells. Crosslinked protein complexes can be detected by decreased mobility on SDS-PAGE followed by western blot detection, size exclusion chromatography, sucrose density gradient sedimentation or mass spectrometry.

Resources:

Photoreactive Amino Acid FAQ

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L-Photo-Methionine
Dulbecco's Modified Eagle's Limiting Medium (DMEM-LM)

Sulfo-NHS (N-hydroxysulfosuccinimide) (Thermo Scientific™)

Thermo Scientific Pierce Sulfo-NHS is a chemical modification reagent for converting carboxyl groups to amine-reactive NHS esters for bioconjugation, crosslinking, labeling and immobilization methods.

Features of Sulfo-NHS:

• Efficiency of EDC-mediated coupling is increased in the presence of Sulfo-NHS
• Amine-reactive NHS esters or Sulfo-NHS esters can be made with any carboxyl-containing molecule
• Sulfo-NHS derivatives are usually directly water-soluble (can be added directly to physiologic buffers) and membrane-impermable (can be used for cell surface labeling)
• High-purity, crystalline Sulfo-NHS can be used to create high-quality activated derivatives

Sulfo-NHS (N-hydroxysulfosuccinimide) enables control and modification of carbodiimide crosslinking reactions involving activation of carboxylates (—COOH) for conjugation with primary amines (—NH2). Derivatives are easily synthesized by mixing the Sulfo-NHS with a carboxyl-containing molecule and a dehydrating agent such as the carbodiimide EDC (EDAC). The method is the basis for generating many types of protein labeling reagents, including amine-reactive fluorescent dyes, biotin affinity tags and pegylation compounds.

Applications:
• Improve efficiency of EDC coupling reactions
• Convert carboxyls to amine-reactive Sulfo-NHS esters
• Crosslink proteins to carboxyl-coated beads or surfaces more efficiently
• Activate nanoparticles with amine-reactive Sulfo-NHS esters

Specifications for Sulfo-NHS:
We manufacture N-hydroxysulfosuccinimide to the highest possible specifications to produce the most specific bioconjugates, to ensure the integrity of your data and to provide you with the highest degree of consistency. Each lot of Sulfo-NHS is tested to meet the following minimum specifications:

Purity—Greater than 95% by quantitative NMR (the highest standard for crosslinker purity);
average lot purity is greater than 99%
Solubility—Sample dissolves at 2 mg/mL in deionized water to yield a clear, colorless solution
Identity—IR scan shows only peaks characteristic of N-hydroxysulfosuccinimide

Product References:
Crosslinker Application Guide -- search for recent literature references for this product

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Pierce™ Premium Grade Sulfo-NHS (N-hydroxysulfosuccinimide)
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Sulfo-SBED Biotin Label Transfer Reagent, No-Weigh™ Format (Thermo Scientific™)

The Thermo Scientific Pierce Sulfo-SBED Biotin Label Transfer Reagent is a multifunctional reagent for labeling a purified protein and then covalently transferring the attached biotin tag onto specific interactors of that protein.

Sulfo-SBED is the abbreviation for Sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-(p-azido benzamido)-hexanoamido) ethyl-1,3'-dithioproprionate. It is a heterobifunctional chemical crosslinker capable of covalently attaching to primary amines at one end and to nearly any protein functional group at the other end. Unlike typical crosslinkers, Sulfo-SBED also includes a biotin group and a cleavable disulfide spacer arm. Together these features allow one to sequentially crosslink interacting proteins and transfer the biotin affinity tag from one protein (i.e., a purified "bait" protein) to another (possibly unknown "prey" protein). Label Transfer is a powerful in vitro method for protein interaction discovery. A growing number of publications feature the use of Sulfo-SBED Biotin Label Transfer Reagent to identify previously unknown protein interaction binding partners and to more fully characterize the specific protein binding domains of other protein interactions.

Typical protocol for label transfer experiment:

• Add a few microliters of dissolved Sulfo-SBED Reagent to 0.5-1 mL of purified bait protein in PBS.
• Incubate mixture for 30-120 minutes on ice or at room temperature in the dark.
• Desalt or dialyze (in subdued light) to remove excess non-reacted Sulfo-SBED from the labeled bait protein.
• Add labeled bait protein to cell lysate or other solution containing putative target protein interactors ("prey").
• When interaction complexes have formed, expose the solution to ultraviolet light (365 nm) for several minutes.
• Analyze products by one of several methods:
Western Blotting: Cleave crosslinks in DTT, separate proteins by SDS-PAGE, and detect biotinylated bands by Western blotting with streptavidin-HRP.
Purification and Mass Spec or Sequencing: Affinity-purify biotinylated proteins or peptide fragments following trypsin digestion and perform MS or sequencing to characterize the proteins involved.

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Sulfo-SBED Biotin Label Transfer Kit - Western Blot Application

SDA (NHS-Diazirine) (succinimidyl 4,4'-azipentanoate) (Thermo Scientific™)

Thermo Scientific Pierce SDA (NHS-Diazirine) combines proven NHS-ester and diazirine-based photoreaction chemistries with conjugate amine-containing molecules with nearly any other functional group via long-wave UV-light activation. A 3.9Å spacer arm separates the two photoreactive groups.

Features of SDA (NHS-Diazirine):

Membrane-permeable—suitable for in vivo intracellular protein crosslinking
Heterobifunctional—NHS ester group reacts with primary amines at pH 7 to 9 to form covalent amide bonds; diazirine (azipentanoate) group reacts efficiently with any amino acid side chain or peptide backbone upon activation with long-wave UV light (330-370 nm)
Controllable—two-step chemical crosslinking is activated using common laboratory UV lamps
Easy to use—these crosslinkers are photo-stable under typical laboratory lighting conditions so there is no need to perform experiments in the dark
Better than aryl azides—the diazirine photoreactive group has better photostability in normal light than phenyl azide groups of traditional photoreactive crosslinkers, yet the diazirine group is more efficiently activated by long-wave UV light

Succinimidyl-diazirine (SDA) reagents are a new class of crosslinkers that combine proven amine-reactive chemistry with an innovative and efficient diazirine-based photochemistry for conjugating amine-containing molecules to nearly any other functional group. The SDA crosslinkers include six compounds differing in spacer arm lengths, ability to cleave the crosslinked proteins, and presence or absence of a charged group for membrane permeability. Protein crosslinking is an important technique used to understand protein structure and to stabilize protein-protein interactions, and these SDA reagents extend the efficiency and range of interactions that can be explored by this approach.

Typical bifunctional amine- or sulfhydryl-reactive crosslinkers require specific amino acid groups (e.g., lysine or cysteine) at the correct distance on both proteins to capture protein interactions. The SDA crosslinkers circumvent this limitation by allowing specific labeling of one protein using the amine-reactive N-hydroxysuccinimide (NHS) ester followed by UV-activated crosslinking via the diazirine moiety to any amino acid side chain or peptide backbone of a second protein. Diazirine-based photocrosslinkers have better photostability than phenyl azide-based photocrosslinkers and are easily activated with long-wave UV light (330-370nm).

Multiple Diazirine Crosslinkers Available
The NHS-ester diazirine derivatives (SDA, LC-SDA and SDAD) lack a charged group and are membrane-permeable. This property makes them ideal for intracellular and intramembrane conjugations. By contrast, Sulfo-SDA, Sulfo-LC-SDA and Sulfo-SDAD contain negatively charged sulfate groups that improve their water solubility and reduce cell membrane permeability, enabling their use for extracellular protein crosslinking. SDAD and Sulfo-SDAD also have a disulfide bond within the spacer that can be cleaved with reducing agents.

• Short (3.9Å) and long (12.5Å) spacer arm varieties
• Membrane-permeable (NHS) and impermeable (Sulfo-NHS) varieties
• Non-cleavable and cleavable (disulfide spacer) varieties

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SM(PEG)4 (PEGylated SMCC crosslinker) (Thermo Scientific™)

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.

Features of SM(PEG)4:

Reactive groups: NHS ester and maleimide
Reactive toward: amino and sulfhydryl groups
• Irreversibly crosslink proteins or peptides by flexible PEG spacer arms
• Polyethylene glycol spacer arms help maintain conjugate solubility
• Pure compound with defined structure and molecular weight, ensuring reproducible protein-modification effects
• PEG spacer provides unique advantages, including increased stability, reduced tendency toward aggregation and reduced immunogenicity
• Ideal for performing controlled, two-step protein conjugations (see Sulfo-SMCC)

SM(PEG)n NHS- and maleimide-activated PEG compounds for crosslinking between primary amines (NH2) and sulfhydryl (SH) groups in proteins and other molecules. A complete set of compounds are offered, each having the same heterobifunctional structure (NHS-PEGn-Maleimide) but differing in the number of discrete ethylene glycol units ( n = 2, 4, 6, 8, 12 or 24). The N-hydroxysuccinimide ester (NHS) group reacts specifically and efficiently with lysine and N-terminal amino groups at pH 7-9 to form stable amide bonds. The maleimide group reacts with reduced sulfhydryls at pH 6.5-7.5 to form stable thioether bonds.

Alternative Names for SM(PEG)n:
• NHS-dPEG-Mal
• Mal-PEG NHS ester
• NHS-PEO-Maleimide
• Succinimidyl-dPEG-maleimide
• Succinimide-Maleimide PEG

Why use crosslinkers with polyethylene glycol (PEG) spacer arms?
PEG-containing reagents have been used to modify proteins to provide specific advantages. Protein PEGylation can improve the stability of the modified protein, protect it from proteolytic digestion, increase its half life in a biological application, mask it from causing an immunogenic response, decrease its antigenicity or potential toxicity, improve its solubility, diminish the potential for aggregation, and minimize interference for both in vitro and in vivo applications. Polyethylene glycol has these effects because it is nontoxic, nonimmunogenic, hydrophilic, water soluble and highly flexible.

Advantages of discrete-length polyethylene glycol compounds:
These reagents are specially synthesized, resulting in homogeneous compounds of defined molecular weight, characterized by discrete chain lengths, providing a greater ability to optimize and characterize surface protein modifications. Typical preparations of PEG compounds are a heterogeneous mixtures composed of a distribution of chain lengths with a specified average molecular weight or approximate number of PEG subunits.

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L-Photo-Methionine (Thermo Scientific™)

Thermo Scientific Pierce L-Photo-Methionine is an analog of L-methionine that can be incorporated into proteins during synthesis and then light-activated to covalently crosslink interacting proteins in cells.

Features of L-Photo-Methionine:

In vivo labeling—incorporate photo-reactive group into proteins using normal cellular machinery of live cells
In vivo crosslinking—find interacting proteins in the native cellular environment
Increased specificity—crosslink interacting proteins correctly positioned at their interfaces within protein interaction domains
Efficient recovery—90% protein recovery in cell lysates after crosslinking
Compatible—crosslink proteins expressed in a wide variety of cell lines, including HeLa, 293T, COS7, U2OS, A549, A431, HepG2, NIH 3T3 and C6
Easy to use—reagents are photo-stable under normal laboratory lighting, eliminating the need to work in the dark

Thermo Scientific L-Photo-Methionine is an analog of L-methionine that can be endogenously incorporated into the primary sequence of proteins during synthesis and then ultraviolet light (UV)-activated to covalently crosslink proteins within protein-protein interaction domains in their native environment within live cells. The powerful method enables both stable and transient protein interactions in cells to be studied and characterized without the use of completely foreign chemical crosslinkers and associated reagent solvents during the interaction experiment that can adversely aspects of the cell biology being studied.

When used in combination with specially formulated limiting media that is devoid of methionine, the photo-activatable derivative is treated like a naturally occurring amino acid by the protein synthesis machinery within the cell. As a result, it can be substituted for methionine in the primary sequence of proteins during catabolism and growth. Photo-methionine derivatives contain diazirine rings that activate when exposed to UV light to become reactive intermediates that form covalent bonds with nearby protein side chains and backbones. Naturally associating binding partners within the cell can be instantly trapped by photoactivation of the diazirine-containing proteins in the cultured cells. Crosslinked protein complexes can be detected by decreased mobility on SDS-PAGE followed by western blot detection, size exclusion chromatography, sucrose density gradient sedimentation or mass spectrometry.

Resources:

Photoreactive Amino Acid FAQ

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L-Photo-Leucine
Dulbecco's Modified Eagle's Limiting Medium (DMEM-LM)

DFDNB (1,5-difluoro-2,4-dinitrobenzene) (Thermo Scientific™)

Thermo Scientific Pierce DFDNB is 1,5-difluoro-2,4-dinitrobenzene, an aryl halide compound with two reactive fluorine atoms that can couple to amine-containing molecules, yielding stable arylamine bonds.

Features of DFDNB:

Reactive groups: aryl halide (both ends)
Reactive towards: amino groups (primary amines)
• Short distance crosslinks

Product References
Crosslinker Application Guide -- search for recent literature references for this product

EZ-Link™ Maleimide-PEG2-Biotin, No-Weigh™ Format (Thermo Scientific™)

Thermo Scientific EZ-Link Maleimide-PEG2-Biotin is a mid-length, maleimide-activated, sulfhydryl-reactive biotinylation reagent that contains a 2-unit ethylene glycol in its spacer arm for increased water-solubility characteristics.

Features of EZ-Link Maleimide-PEG2-Biotin:

Protein labeling—biotinylate antibodies or other proteins for use in protein methods
Thiol-reactive—reacts with sulfhydryls (-SH), such as the side-chain of cysteine (C)
Maleimide-activated—perform reactions at pH 6.5 to 7.5 in buffers such as PBS
Pegylated—spacer arm contains a hydrophilic, 2-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—can be dissolved directly in aqueous buffers for labeling reactions
Medium length—spacer arm (total length added to target) is 29.1 angstroms

Maleimide-PEG2-Biotin enables simple and efficient biotinylation of antibodies, cysteine-containing peptides and other thiol-containing molecules. The maleimide group reacts specifically and efficiently with reduced thiols (sulfhydryl groups,—SH) at pH 6.5 to 7.5 to form stable thioether bonds. The hydrophilic, 2-unit 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 segment adds length and flexibility to the spacer arm, minimizing 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. Three types of sulfhydryl-reactive compounds are available: maleimido, iodoacetyl and pyridyldithiol. Maleimide reagents specifically react with sulfhydryl groups (-SH) in near-neutral buffers to form permanent thioether bonds.

In proteins, sulfhydryls exist where there are cysteine (C) residues. Cystine disulfide bonds must be reduced to make sulfhydryl groups available for labeling. Hinge-region disulfide bridges of antibodies can be selectively reduced to make functional half-antibodies that can be labeled.

NHS-Azide (Thermo Scientific™)

Thermo Scientific NHS-Azide is an amine-reactive compound that can be used to derivatize primary amines of proteins or amine-coated polymer surfaces for ligation to phosphine-modified molecules.

This NHS-ester compound reacts to form covalent bonds with primary amines (e.g., side chain of lysine residues or aminosilane-coated surfaces). The azide (N3) group reacts with phosphine-labeled molecules by a mechanism known as Staudinger chemistry, enabling efficient and specific conjugation of derivatized molecules in biological samples.

Features of NHS-Azide:

Soluble—reagent easily dissolves in water-miscible solvents 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
Specific—NHS esters are specific for covalent attachment to primary amines (side chain of lysine or N-terminus of polypeptides)
Chemoselective—azide and phosphine groups do not react or interfere with components of biological samples (bioorthogonal) but conjugate to one another with high efficiency

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NHS-PEG4-Azide
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Pierce™ Controlled Protein-Protein Crosslinking Kit (Thermo Scientific™)

The Thermo Scientific Pierce Controlled Protein-Protein Crosslinking Kit contains essential crosslinkers, modification reagents and buffers for conjugating antibodies, enzymes and other purified proteins needed as probes.

This crosslinking kit contains everything you need to help you insert an unbreakable molecular bridge between practically any two purified proteins and do it successfully right out of the box.

Features of the Controlled Protein-Protein Crosslinking Kit:

• Easy-to-control, stepwise coupling of two proteins—You are in charge of the chemistry. Stop and restart at convenient points or go to completion. It is your choice.
• Better protocol…logically presented and clearly worded instructions which will be appreciated by novice and expert—Instructions teach the method and chemistry of this crosslinking strategy to the user while providing a high likelihood of success. Great for life scientists attempting to prepare a conjugate or crosslink two proteins for the first time.
• Reliable non-cleavable heterobifunctional crosslinking agent—A very popular choice for a crosslinking agent was selected for the kit offering proven chemistry ,highly stabile intermediates and efficient formation of the target conjugate adding to the ease of use and likelihood of success.
• All reagents necessary to affect the crosslinking—Cuts costs over purchasing "catalog size" packaging of all the components by more than 60%. If it's not in the kit, you don't need it!
• Includes disulfide (S-S) reductants and thiolation reagents—Allows for all possibilities regarding the state of the—SH group on one of the proteins to be coupled.
• Ellman's Reagent colorimetric—SH group determination chemistry built into the protocols—You have the option to see how well your reactions went. eg. determine the extent of—SH introduction or estimate the incorporation of maleimide reactive group.

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NHS-PEG4-Azide (Thermo Scientific™)

Thermo Scientific NHS-PEG4-Azide is an amine-reactive compound that can be used to derivatize primary amines of proteins or amine-coated polymer surfaces for ligation to phosphine-modified molecules.

This NHS-ester compound reacts to form covalent bonds with primary amines (e.g., side chain of lysine residues or aminosilane-coated surfaces). The azide (N3) group reacts with phosphine-labeled molecules by a mechanism known as Staudinger chemistry, enabling efficient and specific conjugation of derivatized molecules in biological samples.

Features of NHS-PEG4-Azide:

Soluble—reagent easily dissolves in water-miscible solvents 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
Specific—NHS esters are specific for covalent attachment to primary amines (side chain of lysine or N-terminus of polypeptides)
Chemoselective—azide and phosphine groups do not react or interfere with components of biological samples (bioorthogonal) but conjugate to one another with high efficiency

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NHS-Azide
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BMPH (N-β-maleimidopropionic acid hydrazide) (Thermo Scientific™)

Thermo Scientific Pierce BMPH is a short, maleimide-and-hydrazide crosslinker for conjugating sulfhydryls (cysteines) to carbonyls (aldehyde or ketones, such as those formed by oxidation of glycoprotein carbohydrates).

Features of BMPH:

Reactive groups: maleimide and hydrazide
Reactive towards: sulfhydryl groups and carbonyl (aldehyde) groups
• Short (8.1A), sulfhydryl-to-aldehyde crosslinker with simple spacer arm (noncleavable)
• Maleimide group reacts with sulfhydryl groups to form stable thioether linkages
• Hydrazide group conjugates to oxidized sugars of glycoproteins and carbohydrates
• Use sodium meta-periodate to oxidize glycosylation (e.g., sialic acid) to reactive aldehyde groups
• Use with EDC to conjugate primary amine of hydrazide group to carboxyl groups

Product References:

Crosslinker Application Guide -- search for recent literature references for this product

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EMCH
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KMUH