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DSS (disuccinimidyl suberate) Thermo Scientific™

Thermo Scientific Pierce DSS, also called disuccinimidyl suberate, is noncleavable and membrane permeable crosslinker that contains an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of an 8-carbon spacer arm.

NHS esters react with primary amines at pH 7-9 to form stable amide bonds, along with release of the N-hydroxysuccinimide leaving group. Proteins, including antibodies, generally have several primary amines in the side chain of lysine (K) residues and the N-terminus of each polypeptide that are available as targets for NHS-ester crosslinking reagents. DSS is first dissolved in an organic solvent such as DMF or DMSO, then added to the aqueous crosslinking reaction. BS3, the water soluble analog of DSS is also available for applications that require a hydrophilic crosslinker (e.g. to effect cell-surface crosslinking). DSS and BS3 have essentially identical crosslinking activity toward primary amines.

Features of disuccinimidyl suberate:

Reactive groups: NHS ester (both ends)
Reactive towards: amino groups (primary amines)
• Amine reactive sulfo-NHS ester reacts rapidly with any primary amine-containing molecule
• Membrane-permeable, allowing for intracellular crosslinking
• High purity, crystalline reagent can be used to create high-purity conjugates
• Non-cleavable
• Water-insoluble (dissolve first in DMF or DMSO); compare to BS3 (Sulfo-DSS)

Applications:
• Chemical crosslinking of intracellular proteins prior to cell lysis and immunoprecipitation
• 'Fix' protein interactions to allow identification of weak or transient protein interactions
• Protein crosslinking to create bioconjugates via single-step reactions
• Immobilize proteins onto amine-coated surfaces

Properties of DSS
• Alternative names: Disuccinimidyl suberate
• Molecular formula: C16H20N2O8
• Molecular weight: 368.35
• Spacer arm length: Å (8 atoms)
• CAS number: 68528-80-3
• Reactive groups: NHS esters, react with primary amines at pH 7.0–9.0

DSS Specifications
We manufacture DSS 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 DSS crosslinker is tested to meet the following minimum specifications:
Identity: IR scan shows only peaks characteristic of the structure and functional groups of DSS
Purity: > 90% by quantitative NMR (the highest standard in crosslinking purity)
Solubility: > 9.2 mg/mL in DMF and DMSO, clear and colorless solution

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

Related Products
BS3 (bis(sulfosuccinimidyl)suberate)

Pierce™ SMCC, No-Weigh™ Format Thermo Scientific™

Thermo Scientific Pierce SMCC is a hetero-bifunctional crosslinker that contain N-hydroxysuccinimide (NHS) ester and maleimide groups that allow covalent conjugation of amine- and sulfhydryl-containing molecules. NHS esters react with primary amines at pH 7–9 to form amide bonds, while maleimides react with sulfhydryl groups at pH 6.5–7.5 to form stable thioether bonds. In aqueous solutions, NHS ester hydrolytic degradation is a competing reaction whose rate increases with pH. The maleimide group is more stable than the NHS-ester group, but will slowly hydrolyze and lose its reaction specificity for sulfhydryls at pH values > 7.5. For these reasons, conjugations with these crosslinkers are usually performed at pH 7.2–7.5, with the NHS ester (amine-targeted) reacted before or simultaneous with the maleimide (sulfhydryl-targeted) reaction.

The cyclohexane ring in the spacer arm of this reagent decreases the rate of hydrolysis of the maleimide group compared to similar reagents that do not contain this ring. This feature enables proteins that have been maleimide-activated with SMCC to be lyophilized and stored for later conjugation to a sulfhydryl-containing molecule. Many maleimide-activated protein products are produced in this manner.

Thermo Scientific No-Weigh products are specialty reagents provided in a pre-aliquoted format. The pre-weighed packaging prevents the loss of reagent reactivity and contamination over time by eliminating the repetitive opening and closing of the vial. The format enables use of a fresh vial of reagent each time, eliminating the hassle of weighing small amounts of reagents and reducing concerns over reagent stability.

Features of SMCC:

Reactive groups: NHS ester and maleimide
Reactive toward: amino and sulfhydryl groups
• Non-cleavable
• Water-insoluble (dissolve first in DMF or DMSO); compare to Sulfo-SMCC
• High purity, crystalline reagent can be used to create high-purity maleimide-activated derivatives
• Cyclohexane bridge confers added stability to the maleimide group making SMCC an ideal crosslinking agent for maleimide activation of proteins. Maleimide groups are stable for 64 hours in 0.1 M sodium phosphate buffer, pH 7 at 4°C.

Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) is a non-cleavable and membrane permeable crosslinker. It contains an amine-reactive N -hydroxysuccinimide (NHS ester) and a sulfhydryl-reactive maleimide group. NHS esters react with primary amines at pH 7-9 to form stable amide bonds. Maleimides react with sulfhydryl groups at pH 6.5-7.5 to form stable thioether bonds. The maleimide groups of SMCC and Sulfo-SMCC and are unusually stable up to pH 7.5 because of the cyclohexane bridge in the spacer arm.

Applications:
• Enzyme labelling of antibodies - both enzyme and antibody specificity can be preserved
• Create specific bioconjugates via one- or two-step crosslinking reactions
• Create sulfhydryl-reactive, maleimide-activated carrier proteins for coupling haptens

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

• Identity: IR scan shows only peaks characteristic of the structure and functional groups of SMCC cross linker
• Purity: ≥ 92% by NMR

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

Pierce™ Premium Grade Sulfo-NHS (N-hydroxysulfosuccinimide) Thermo Scientific™

Thermo Scientific Pierce Premium Grade Sulfo-NHS is our highest quality formulation of this N-hydroxysuccinimide chemical modification reagent, specially characterized for applications where product integrity and risk minimization are paramount.

Features of Premium Grade Sulfo-NHS:

Sulfo-NHS—use with EDC crosslinker to prepare amine-reactive NHS ester reagents
High quality—identity and purity confirmed by several tests, including quantitative NMR
Product integrity—enhanced level of testing and characterization compared to standard grade
Lot retention—ample supply of past lots retained to ensure future process testing
Change management—Change Control Notification (CCN) service
Consistent manufacture—batch-specific manufacturing documentation review

Applications:
• Improve efficiency of EDC coupling reactions
• Convert carboxyls to amine-reactive sulfo-NHS esters
• Membrane-impermable for cell surface labeling
• Crosslink proteins to carboxyl-coated beads or surfaces more efficiently
• Activate nanoparticles with amine-reactive sulfo-NHS esters

Compared to the standard grade product, Premium Grade Sulfo-NHS provides more clearly defined quality and product support by including (a) increased analytical testing and product characterization, (b) greater batch-specific information and quality assurance review, (c) extensive lot sample retention and (d) change control notification. 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.

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

Related Products
Sulfo-NHS (N-hydroxysulfosuccinimide)
Pierce™ Premium Grade EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)

Pierce™ Sulfo-SANPAH, No-Weigh™ Format Thermo Scientific™

Thermo Scientific Pierce Sulfo-SANPAH is a hetero-bifunctional crosslinker that contains an amine-reactive N-hydroxysuccinimide (NHS) ester and a photoactivatable nitrophenyl azide. NHS esters react efficiently with primary amino groups (-NH2) in pH 7–9 buffers to form stable amide bonds. The reaction results in the release of sulfo-N-hydroxy-succinimide. When exposed to UV light, nitrophenyl azides form a nitrene group that can 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). Sulfo-SANPAH, supplied as a sodium salt, is useful for cell-surface protein crosslinking and possesses charged groups for water solubility to a concentration of 10 mM.

Thermo Scientific No-Weigh products are specialty reagents provided in a pre-aliquoted format. The pre-weighed packaging prevents the loss of reagent reactivity and contamination over time by eliminating the repetitive opening and closing of the vial. The format enables use of a fresh vial of reagent each time, eliminating the hassle of weighing small amounts of reagents and reducing concerns over reagent stability.

Features of Sulfo-SANPAH:

Reactive groups: sulfo-NHS ester and nitrophenyl azide
Reactive towards: amino groups and any nucleophile
• Non-cleavable
N-Sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate
• Optimal photolysis occurs at 320-350 nm; minimizes damage to biomolecules by irradiation
• Water soluble; non-cleavable

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

SM(PEG)8 (PEGylated, long-chain 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)8:

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.

Related Products
SM(PEG)6 (PEGylated, long-chain SMCC crosslinker)
SM(PEG)12 (PEGylated, long-chain SMCC crosslinker)
SM(PEG)24 (PEGylated, long-chain SMCC crosslinker)

TSAT (tris-(succinimidyl)aminotriacetate) Thermo Scientific™

Thermo Scientific Pierce TSAT is tris-(succinimidyl) aminotriacetate, a novel trifunctional crosslinker that contains three short spacers arms linked to a central nitrogen atom, each ending in an amine-reactive NHS-ester group.

Features of TSAT:

Reactive groups: NHS ester (three ends)
Reactive towards: amino groups (primary amines)
• NHS ester couples to amines at pH 7-9 form stable amide bonds
• Trifunctional amine-reactive crosslinking agent
• Non-cleavable
• Water-insoluble (dissolve first in DMF or DMSO)

Applications:
• Preparation of multicomponent aggregates
• Polyerization of protein interactions for nearest-neighbor studies
• Core molecule for the construction of dendritic polymers

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

Related Products
TSAT (tris-(succinimidyl)aminotriacetate)

Pierce™ BS(PEG)5, No-Weigh™ Format Thermo Scientific™

BS(PEG)9 is a bis-succinimide ester-activated PEG compound used for crosslinking between primary amines (—NH2) in proteins and other molecules. The N-hydroxysuccinimide ester (NHS) groups at either end of the PEG9 spacer react specifically and efficiently with lysine and N-terminal amino groups at pH 7-9 to form stable amide bonds.

Features of BS(PEG)9:

Reactive groups: sulfo-NHS ester (both ends)
Reactive towards: amino groups (primary amines)
• NHS esters react with—NH2 groups at pH 7-9, forming stable amide bonds
• Water-soluble; compare to non-pegylated BS3 (also soluble) and DSS (water-insoluble)
• Membrane-impermeable, allowing for cell surface labeling
• 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 small molecule or peptide conjugations

Alternative Names for BS(PEG)n:

• Bis-NHS-dPEG
• NHS-PEG-NHS
• BS(PEO)5
• Bis(NHS)PEO5
• BS-PEO9
• dPEG NHS ester
• Bis-succinimide ester PEG 400

Product References
Crosslinker Application Guide -- search for recent literature references for this product
Bioconjugate Techniques, Academic Press (Part No. 20036)

Related Products
BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate)
BS3 (bis(sulfosuccinimidyl)suberate)

Pierce™ Premium Grade Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) Thermo Scientific™

Thermo Scientific Pierce Premium Grade Sulfo-SMCC is our highest quality formulation of amine-to-sulfhydryl crosslinker, specially characterized for applications where product integrity and risk minimization are paramount.

Compared to the standard grade product, Premium Grade Sulfo-SMCC provides more clearly defined quality and product support by including (a) increased analytical testing and product characterization, (b) greater batch-specific information and quality assurance review, (c) extensive lot sample retention and (d) change control notification. Sulfo-SMCC is a water-soluble, non-cleavable and membrane-impermeable amine-to-sulfhydryl crosslinker that contains NHS-ester and maleimide reactive groups at opposite ends of a medium-length cyclohexane spacer arm (8.3 angstroms). NHS esters react with primary amines at pH 7-9 to form stable amide bonds. Maleimides react with sulfhydryl groups at pH 6.5-7.5 to form stable thioether bonds. The maleimide groups of Sulfo-SMCC are unusually stable up to pH 7.5 because of the cyclohexane bridge in the spacer arm. Because it contains the hydrophilic sulfonyl moiety, Sulfo-SMCC is soluble up to nearly 10 mM in water and many commonly used buffers, thus avoiding the use of organic solvents which may perturb protein structure.

Features of Sulfo-SMCC:

Sulfo-SMCC—a water-soluble amine-to-sulfhydryl crosslinker with a stabilizing cyclohexane spacer arm
High quality—identity and purity confirmed by several tests, including quantitative NMR
Product integrity—enhanced level of testing and characterization compared to standard grade
Lot retention—ample supply of past lots retained to ensure future process testing
Change management—Change Control Notification (CCN) service
Consistent manufacture—batch-specific manufacturing documentation review

Applications:
• Enzyme labelling of antibodies - both enzyme and antibody specificity can be preserved
• Create specific bioconjugates via one- or two-step crosslinking reactions
• Create sulfhydryl-reactive, maleimide-activated carrier proteins for coupling haptens

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

Related Products
Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate)

Sulfo-SBED Biotin Label Transfer Reagent 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.

Related Products
Sulfo-SBED Biotin Label Transfer Kit - Western Blot Application

Pierce™ Premium Grade EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) Thermo Scientific™

Thermo Scientific Pierce Premium Grade EDC is our highest quality formulation of this popular carbodiimide crosslinker, specially characterized for applications where product integrity and risk minimization are paramount.

Features of Premium Grade EDC:

EDC—carbodiimide crosslinker for conjugation between carboxylates and amines
High quality—identity and purity confirmed by several tests
Product integrity—enhanced level of testing and characterization compared to standard grade
Lot retention—ample supply of past lots retained to ensure future process testing
Change management—Change Control Notification (CCN) service
Consistent manufacture—batch-specific manufacturing documentation review

Compared to the standard grade product, Premium Grade EDC provides more clearly defined quality and product support by including (a) increased analytical testing and product characterization, (b) greater batch-specific information and quality assurance review, (c) extensive lot sample retention and (d) change control notification. EDC is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, a water-soluble carbodiimide crosslinker that activates carboxyl groups for spontaneous reaction with primary amines, enabling peptide immobilization and hapten-carrier protein conjugation. In the presence of N-hydroxysulfosuccinimide (sulfo-NHS), EDC can be used to convert carboxyl groups to amine-reactive sulfo-NHS esters. This is accomplished by mixing the EDC with a carboxyl containing molecule and adding sulfo-NHS.

Specifications of Premium Grade EDC
We manufacture crosslinking reagents to ensure the highest possible overall product integrity, consistency and performance for the intended research applications.
• Visual: White crystalline powder, free of foreign matter
• Identity: IR scan shows only peaks characteristic of EDC
• Water content: Karl Fischer titration, ≤ 2%
• Trace metals: All metals, including lead, ≤ 50 ppm
• Elemental analysis: Reported values for C, H, and N

Applications:
• Conjugate carboxyl and amino groups among peptides and proteins
• Couple haptens to immunogenic carrier proteins (e.g., attach a peptide to KLH)
Immobilize peptide antigens to affinity-purify antibodies
• Create NHS-activated, amine-reactive labeling compounds
• Crosslink proteins to carboxyl coated beads or surfaces
• Activate nanoparticles with amine-reactive sulfo-NHS esters

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

Related Products
EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)
Pierce™ Premium Grade Sulfo-NHS (N-hydroxysulfosuccinimide)

SBAP (succinimidyl 3-(bromoacetamido)propionate) Thermo Scientific™

Thermo Scientific Pierce SBAP is a short (6.2 angstrom) crosslinker for amine-to-sulfhydryl conjugation via N-hydroxysuccinimide (NHS) ester and bromoacetyl reactive groups.

Features of SBAP:

Reactive groups: NHS ester and bromoacetyl
Reactive toward: amino and sulfhydryl groups
• NHS ester reacts with primary amines at pH 7-9 to form a stable amide bond
• Bromacetyl group reacts with sulfhydryl groups at pH > 7.5 to form stable thioether bonds
• Non-cleavable
• Water-insoluble (dissolve first in DMF or DMSO)
• Spacer maintains peptide-like character in the crosslinked species
• Resulting crosslink is susceptible to acid hydrolysis
• Useful for preparing cyclic peptides and peptide conjugates

Product References:

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

BS2G-d0 (bis(sulfosuccinimidyl) glutarate-d0) Thermo Scientific™

Thermo Scientific Pierce BS2G-d0/d4 reagents form a matched set of heavy (deuterated) and light mass varieties of a short, amine-reactive crosslinker that facilitates the identification of crosslinked peptides by mass spectrometry.

The BS2G-d0/d4 reagents are mass-analogs of BS2G crosslinker. The d-zero variety (BS2G-d0) possesses ordinary hydrogen atoms while the d-four variety (BS2G-d4) contains four deuterium atoms that make it exactly four hydrogen mass units heavier. When the two varieties are mixed and used to crosslink interacting proteins, distinctive mass patterns result that enable unambiguous identification of the crosslinked molecules.

Highlights:

Deuterium-labeled BS2G and its standard hydrogen-only analog—specially manufactured for crosslinking protein structure:function analysis by mass spectrometry
Easy identification of crosslinked fragments—peptide masses differing by 4 units unambiguously identify crosslink at specific loci within a protein interaction structure
Application-specific instructions—help guide the first time user through the application
Method requires only microgram amounts of protein—excellent alternative to NMR or X-ray crystallography-based methods that require large amounts of protein, special solvents or crystal formation

Addition of an equimolar mixture of two identical crosslinking agents that differ only in the number of deuterium atoms in their chemical structures provides a powerful method to identify low-abundance, singly-crosslinked peptides by mass spectrometry. Crosslinking protein interactions with BS2G-d0/d4 reagents generates distinctive mass patterns in which crosslinked peptides differ by 4 mass units after enzymatic digestion of the crosslinked protein or protein complex. Further analysis of the reaction products can yield low resolution three dimensional structure information.

BS2G-d0/d4 and BS3-d0/BS3-d4 reagents are homobifunctional, amine-reactive, non-cleavable, water-soluble crosslinking agents with defined spacer arm lengths. These reagents can act as molecular rulers for estimation of spatial relationships in protein structure-function studies. Both light and deuterated analogs react efficiently with primary amine (—NH2) at pH 7-9 to form stable amide bonds. Proteins generally contain several primary amines in the side chain of Lysine (K) residues and the N-terminus of each polypeptide that are available as targets for the two sulfo-NHS ester reactive groups found in each of these reagents. These reagents are supplied as a sodium salt and are soluble in water at a concentration up to 10 mM.

Related Products
BS2G-d4 (bis(sulfosuccinimidyl) 2,2,4,4-glutarate-d4)

EZ-Link™ Sulfo-NHS-SS-Biotin, No Weigh Format Thermo Scientific™

Thermo Scientific EZ-Link Sulfo-NHS-SS-Biotin is a water-soluble, NHS-ester biotinylation reagent whose spacer arm includes a cleavable disulfide bond for reversible labeling of proteins and cell surface primary amines.

Features of EZ-Link Sulfo-NHS-SS-Biotin:

Protein labeling – biotinylate antibodies to facilitate immobilization, purification or detection
Cell surface labeling – biotinylates only surface proteins of whole cells because the negatively charged reagent does not permeate cell membranes
Amine-reactive—reacts with primary amines (-NH2), such as lysine side-chains, or the amino-termini of polypeptides
Cleavable—disulfide bond in spacer arm allows the biotin label to be removed using reducing agents such as DTT; only a small sulfhydryl group remains attached to the molecule
Soluble – charged sulfo-NHS group increases reagent water solubility compared to ordinary NHS-ester compounds
Medium length—spacer arm (total length added to target) is 24.3 angstroms; it consists of the native biotin valeric acid group extended by a 7-atom chain

Sulfo-NHS-SS-Biotin is a thiol-cleavable amine-reactive biotinylation reagent that contains an extended spacer arm to reduce steric hindrances associated with avidin binding. This reagent is water-soluble, enabling biotinylation to be performed in the absence of organic solvents such as DMSO or DMF for applications that cannot tolerate solvents or are complicated by their inclusion. The compound is particularly useful for labeling and purifying cell surface proteins, because (a) its sulfonate group prevents it from permeating cell membranes and (b) its cleavable spacer arm enables initially biotinylated proteins to be released from streptavidin affinity columns.

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™ Sulfo NHS-SS Biotinylation Kit
EZ-Link™ Micro Sulfo-NHS-SS-Biotinylation Kit
Pierce™ Premium Grade Sulfo-NHS-SS-Biotin

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.

Related Products
Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate)

EZ-Link™ Phosphine-PEG4-Desthiobiotin, No-Weigh™ Format Thermo Scientific™

Thermo Scientific EZ-Link Phosphine-PEG4-Desthiobiotin is a pegylated reagent whose biotin-like group is elutable from streptavidin, activated for chemoselective ligation and purification of azide targets tagged by metabolic labeling.

Features of EZ-Link Phosphine-PEG4-Desthiobiotin:

Desthiobiotin—analog of biotin allows easy elution from streptavidin, an ideal feature for affinity purification applications
Chemoselective—the phosphine reactive group is specific in biological samples for azide-tagged molecules, ensuring specific capture and purification of labeled targets
Compatible—Staudinger reaction chemistry proceeds efficiently in simple buffer conditions; requires no accessory reagents such as copper or reducing agents
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
Soluble—easily dissolves in water-miscible solvents (e.g., DMSO) for subsequent dilution in aqueous reaction mixtures with cell lysates and other biological samples

When used in combination with azide labeling strategies, Phosphine-PEG4-Desthiobiotin enables affinity purification of protein interactions and post-translational modifications using streptavidin resins. The phosphine moiety selectively crosslinks via the Staudinger reaction mechanism to molecules that have been tagged or metabolically labeled with bioorthogonal azide groups. The desthiobiotin tag binds to streptavidin and other biotin-binding proteins with high specificity yet readily elutes with mild conditions (i.e., by competitive displacement with regular, free biotin). As such, this reagent is a useful alternative to Phosphine-PEG3-Biotin (Part No. 88901) for avidin-biotin techniques in which nondenaturing elution of the labeled proteins is desired. The No-Weigh Format (5 x 1 mg resealable vials) eliminates the difficulties associated with weighing small quantities of reagent and maximizes protection of unused reagent from degradation.

Desthiobiotin vs. Biotin
Desthiobiotin is a single-ring, sulfur-free analog of biotin that binds to streptavidin with nearly equal specificity but less affinity than biotin (1/Kd = 1011 vs. 1015M, respectively). Consequently, desthiobiotinylated bait proteins and their interacting partners can be eluted readily and specifically from streptavidin affinity resin using mild conditions based on competitive displacement with free biotin. For pull-down assay experiments with biological samples, this soft-release characteristic of desthiobiotin also helps to minimize co-purification of endogenous biotinylated molecules, which remain bound to streptavidin upon elution of the target protein complexes with free biotin. The modified avidin-biotin affinity system also eliminates the need to use harsh elution conditions that might disassociate complexes and/or damage the target protein or cell. Desthiobiotin-based techniques are ideal when using native or recombinant proteins that are not expressed with a fusion tag and when isolating captured proteins under native conditions, such as targeting intact cells or cell surface proteins.

Labeling with Phosphine Reagents
Azide groups can be introduced into proteins or other cellular targets through in vivo labeling with azide-derivatives of naturally occurring metabolic building blocks. Because phosphines and azides are foreign and invisible to biological systems (bioorthogonal), they comprise a chemoselective (mutually specific) ligation pair for labeling and conjugation.
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