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Pierce™ Asp-N Protease, MS Grade Thermo Scientific™

Thermo Scientific Asp-N Protease, MS Grade, is a highly specific endoproteinase used to improve sequence coverage in mass spectrometry protein identification applications.

Features of MS Grade Asp-N Protease:

• Complementary to tryptic digests—hydrolyzes proteins specifically at the amino side of aspartate and cysteic acid residues
• Increased sequence coverage—better protein characterization results from overlapping peptides with complementary chromatographic, ionization and fragmentation properties
• High specific activity—greater than 20,000 units/mg protein
• N-terminal arginine cleavage specificity—at least 90% for a complex protein sample
• Stable—provided in a lyophilized format

Product Details
This Asp-N is a mass spectrometry (MS)-grade zinc metalloproteinase derived from a mutant strain of Pseudomonas fragi and requires a trace amount of zinc for activity. Asp-N can be used alone or in parallel with trypsin or other proteases to produce protein digests for peptide mapping and protein sequencing. Asp-N protease is suitable for either in-solution or in-gel digestion workflows. This Asp-N enzyme is packaged lyophilized (2 µg).

The endoproteinase AspN cleaves primarily at amino side of aspartate residues and cysteic acid residues that result from the oxidization of cysteine residues, generating a limited number of peptide fragments. Cleavage can also occur at glutamic residues; however, the rate of cleavage at the glutamyl residues is significantly lower than the rate of cleavage at the aspartic acid residues. AspN can efficiently digest protein in 2-20 hours at 37°C. AspN remains active under denaturing conditions such as 1M urea, 2M guanidine·HCl, 0.1% SDS, 2% CHAPS and 10% acetonitrile with optimal activity in the pH range of 6-8. This lyophilized enzyme has a mass of 27 kDa and is stable for 1 year when stored at -20°C.

Applications
• Improved sequence coverage of protein digests
• In-solution digestion of proteins
• In-gel digestion of proteins

Pierce™ Citraconic Anhydride Thermo Scientific™

Thermo Scientific Pierce Citraconic Anhydride reacts with primary amines to to form an amide bond with a terminal carboxyl group. This modification can be reversed using acidic conditions (pH 3-4) or with hydroxylamine.

Citraconic Anhydride (2-methylmaleic anhydride) is used to block primary amine groups at alkaline pH values (pH 7-9). At acidic pH (3-4), the amide linkage is hydrolyzed and citraconic acid is released, revealing the original amine.

Features of Citraconic Anhydride:

• Temporarily block amines to derivatize other parts of the molecule
• Unblock amines using acidic conditions or hydroxylamine

MT(PEG)4 Methyl-PEG-Thiol Compound Thermo Scientific™

Thermo Scientific Pierce MT(PEG)4, or methyl-PEG4-thiol, is a methyl- and sulfhydryl-terminated compound that contains a 4-unit polyethylene glycol (PEG) spacer and is used to modify surfaces such as quantum dots, monolayers and magnetic particles.

Features of MT(PEG)4:

Metal-binding—terminal monodentate thiol reacts spontaneously with gold surfaces through dative binding
Methyl—terminal methyl group is unreactive, the primary function of this compound being to coat quantum dot and other surfaces to reduce nonspecific binding in assays
Polyethylene glycol—PEG groups are flexible, non-immunogenic, hydrophilic, and significantly reduce nonspecific binding of surfaces for protein methods
Spacer arm—four-unit PEG spacer makes compound nearly 16 angstroms long

MT(PEG)4 is monodentate thiol-terminated and methyl pegylation reagent. The PEG compound has a defined molecular weight and spacer length and is useful for modifying surfaces such quantum dots, self-assembled monolayers and magnetic particles. Functionalization of solid surfaces with polyethylene glycol spacers such as this one significantly reduces nonspecific protein binding.

The use of MT(PEG)4 with CT(PEG)12 in surface modification can form a hydrophilic 'lawn' of methyl ether-terminated PEGs with periodic exposed carboxylic acid-containing PEGs. The exposed carboxylic acid groups can be coupled to affinity ligands using the carbodiimide coupling reaction with EDC and sulfo-NHS.

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ML(PEG)4 Methyl-PEG-Lipoamide Compound

NeuCode™ Lysine-080 Thermo Scientific™

Thermo Scientific™ NeuCode™ amino acids augment the level of multiplexing achievable for the metabolic labeling of proteins for mass spectrometry analysis. NeuCode™ Lysine-080 (3,3,4,4,5,5,6,6-D8 L-Lysine-2HCl) may also be used with traditional SILAC to improve flexibility of multiplexing options or to reduce complexity of analysis.

General features of NeuCode SILAC labeling:
• Labeling efficiency—100% label incorporation into proteins of living cells without toxicity
• Compatible—may be multiplexed with existing SILAC amino acids
• Time-saving—not necessary to label to 100% incorporation if only using heavy amino acids
• High-quality supplements—heavy amino acids with >98% isotope purity

Stable isotope labeling with amino acids in cell culture (SILAC) is a powerful method to identify and quantify relative differential changes in complex protein samples. NeuCode metabolic labeling is similar to SILAC, but differs in that the labeling only utilizes heavy amino acids. The increased multiplexing capability of NeuCode amino acids is possible through the use of mass defects from extra neutrons in the stable isotopes. These small mass differences may be resolved on high resolution mass spectrometers (Thermo Scientific™ Orbitrap™ Elite™, Q Exactive™, Orbitrap™ Fusion™ Tribrid™, and Orbitrap™ Fusion™ Lumos™ Tribrid™ mass spectrometers). Use of only heavy amino acids eliminates the need for 100% incorporation of amino acids used for SILAC (both heavy and light), and may be especially useful for studies with primary cells.

NeuCode amino acids are used together with specialized cell culture media that are deficient in essential amino acids. Heavy L-lysine is used for SILAC analysis of peptides that have been digested with trypsin or LysC. NeuCode Lysine-080 may be used with 4,4,5,5-D4 L-lysine or 13C6 15N2 L-lysine for duplex experiments and may be combined with light lysine for three-plex experiments.

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L-Lysine-2HCl, 4,4,5,5-D4 for SILAC
L-Lysine-2HCl, 13C6, 15N2 for SILAC
Fetal Bovine Serum, dialyzed, US origin
DMEM Media for SILAC

NeuCode is a trademark of WARF.

Pierce™ SATA (N-succinimidyl S-acetylthioacetate) Thermo Scientific™

Thermo Scientific Pierce SATA is a short-chain (2.8 angstrom spacer arm) reagent for covalent modification of primary amines and addition of a protected yet exposable sulfhydryl group, enabling heterobifunctional crosslinking strategies.

Features of SATA:

• Adds a protected sulfhydryl that can be deprotected by hydroxylamine
• Allows long-term storage of the sulfhydryl-modified molecule
• Forms cleavable disulfide bonds with other sulfhydryl-containing molecules
• Reacts with primary amines (e.g., lysine residues proteins) to form stable amide bonds
• Preserves protein activity with its mild, non-denaturing reaction conditions

SATA (N-succinimidyl S-acetylthioacetate) adds sulfhydryl groups to proteins and other amine-containing molecules in a protected form. The modified molecule can be stored indefinitely and treated with hydroxylamine to expose the labile sulfhydryl group when needed for conjugation reactions. SATA contains an N-hydroxysuccinimide (NHS) ester, which forms a stable, covalent amide bond with primary amines (i.e., lysine residues and the amino termini of proteins) and releases NHS as a by-product. De-protection (deacylation) to generate a free sulfhydryl is accomplished using hydroxylamine-HCl.

Sulfhydryl groups present on proteins, peptides and other compounds are important in protein chemistry/modification reactions. Frequently, thiols are unavailable or absent within the molecules of interest. Several reagents and techniques are available for introducing sulfhydryl groups or disulfides into proteins and peptides, including Traut's Reagent, variants of SPDP, and variants of SATA.

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Pierce™ Sulfhydryl Addition Kit
Pierce™ SATP
Pierce™ SAT(PEG)4

Pierce™ EMCA (N-ε-maleimidocaproic acid) Thermo Scientific™

Thermo Scientific Pierce EMCA is a carboxylic acid compound with a terminal maleimide group that can modify proteins and other molecules by EDC-mediated and sulfhydryl-targeted conjugation.

Features EMCA:

• Convert sulfhydryl groups to carboxyl groups for conjugation to amines via carbodiimide (EDC)
• Prepare maleimide-activated proteins using EDC to modify primary amines
Maleimides react with—SH groups at pH 6.5-7.5, forming stable thioether linkages

EMCA (N-epsilon-maleimidocaproic acid) is a heterobifunctional crosslinker that reacts with sulfhydryl groups to present a terminal carboxyl group. Additionally, the carboxyl can be coupled to amine-containing molecules using the water-soluble carbodiimide EDC. The EDC reaction results in an intermediate ester, which can react with primary amines to form amide bonds. Thus, amine-containing molecules can be modified to contain sulfhydryl-reactive maleimides.

Pierce™ Sodium meta-Periodate Thermo Scientific™

Thermo Scientific Pierce Sodium meta-Periodate is a gentle oxidizing agent that cleaves cis-diols in carbohydrate sugars to create amine-reactive aldehydes, providing many uses relating to the study and detection of glycoproteins.

Features of Sodium meta-Periodate:

• Convert sugars in sialic acid and other glycosylation groups to reactive aldehydes
Immobilize glycoproteins to a hydrazide-activated solid support
• Conjugate antibodies to glycoprotein enzymes, such as horseradish peroxidase
• Probe for cell-surface polysaccharides
• Detect carbohydrate-containing proteins using hydrazide-containing probes

Sodium meta-periodate converts cis-glycol groups in carbohydrates to amine-reactive aldehyde groups. Carbohydrate groups in glycoproteins are excellent sites for modification or crosslinking reactions because they allow the conjugation reaction to be directed away from amino acids in the polypeptide chain that could be critical for protein activity. Sodium meta-periodate cleaves bonds between adjacent carbon atoms that contain hydroxyl groups (cis-glycols), creating two aldehyde groups that are spontaneously reactive to amine- and hydrazide-activated labeling, immobilization supports and crosslinking reagents.

Dithiothreitol (DTT) Invitrogen™

Disufide crosslinks of cystines in proteins can be reduced to cysteine residues by dithiothreitol (DTT).

CT(PEG)12 Carboxy-PEG-Thiol Compound Thermo Scientific™

Thermo Scientific Pierce CT(PEG)12, or carboxy-PEG12-thiol, is a carboxyl- and sulfhydryl-terminated compound that contains a 12-unit polyethylene glycol (PEG) spacer and is used to modify surfaces such as quantum dots, monolayers and magnetic particles.

CT(PEG)12 is monodentate thiol-terminated and carboxylic acid pegylation reagent. The PEG compound has a defined molecular weight and spacer length and is useful for modifying surfaces such quantum dots, self-assembled monolayers and magnetic particles. Functionalization of solid surfaces with this polyethylene glycol reagent provides carboxylate 'handles' for protein immobilization via EDC crosslinking and significantly reduces nonspecific protein binding and provides.

Features of carboxy-PEG12-thiol:

Metal-binding—terminal monodentate thiol reacts spontaneously with gold surfaces through dative binding
Carboxylate—terminal carboxylic acid group allows conjugation to amine-containing affinity ligands using carbodiimide (EDC) and Sulfo-NHS crosslinking chemistry
Polyethylene glycol—PEG groups are flexible, non-immunogenic, hydrophilic, and significantly reduce nonspecific binding of surfaces for protein methods
Spacer arm—12-unit PEG spacer is nearly 48 angstroms long, minimizing steric hindrance for protein immobilization and binding assays

The use of MT(PEG)4 with CT(PEG)12 in surface modification can form a hydrophilic 'lawn' of methyl ether-terminated PEGs with periodic exposed carboxylic acid-containing PEGs. The exposed carboxylic acid groups can be coupled to affinity ligands using the carbodiimide coupling reaction with EDC and sulfo-NHS.

Related Products
CL(PEG)12 Carboxy-PEG-Lipoamide Compound

Pierce™ Glu-C Protease, MS Grade Thermo Scientific™

Thermo Scientific Glu-C Protease, MS Grade, is a highly specific endoproteinase use to improve sequence coverage in mass spectrometry protein identification applications.

Features of Glu-C Protease, MS Grade:

• Complementary to tryptic digests—hydrolyzes proteins specifically at the carboxyl side of glutamic acids
• Increased sequence coverage—better protein characterization results from overlapping peptides with complementary sequence coverage and chromatographic, ionization and fragmentation properties
• High specific activity—greater than 500 units/mg protein
• N-terminal arginine cleavage specificity—at least 90% for a complex protein sample
• Stable—provided in a lyophilized format

Product Details
This Glu-C is a mass spectrometry (MS)-grade serine protease isolated from Staphylococcus aureus. Glu-C can be used alone or alongside trypsin or other proteases to produce complementary protein digests for peptide mapping and protein sequencing. Glu-C protease is suitable for either in-solution or in-gel digestion workflows. This Glu-C enzyme is packaged lyophilized (5 x 10 µg).

The endoproteinase Glu-C, also referred to as V-8 Protease, specifically cleaves the carboxyl side of glutamic residues when reactions are carried out in ammonium bicarbonate and ammonium acetate buffers, generating a limited number of peptide fragments. Cleavage can also occur at both glutamic and aspartic residues in phosphate buffers. Glu-C can efficiently digest protein in 5-18 hours at 37°C. Glu-C Protease remains active under denaturing conditions such as 2M urea, 1M guanidine·HCl, 0.1% SDS, 2% CHAPS and 20% acetonitrile. Glu-C activity is optimal at pH 8. This lyophilized enzyme has a mass of 27 kDa and is stable for 1 year when stored at -20°C.

Applications
• Improved sequence coverage of protein digests
• In-solution digestion of proteins
• In-gel digestion of proteins

MA(PEG)4 Methyl-PEG-Amine Compound Thermo Scientific™

Thermo Scientific Pierce MA(PEG)4 is a methyl- and amine-terminated polyethylene glycol reagent that is useful for a variety of surface-modification and molecule-pegylation applications.

Features of MA(PEG)4:

• Fully characterized PEGylation reagent with defined PEG chain length; discrete molecular weight for consistency of performance in protein-modification applications
• PEG spacer provides unique advantages, including increased stability, reduced tendency toward aggregation and reduced immunogenicity
• Allows site-specific labeling of primary amines or carboxyl groups on proteins or surfaces
• Easy-to-follow instructions increase the likelihood of a successful outcome

MA(PEG)n is the abbreviation for a set of compounds having polyethylene glycol (PEG) spacers and terminal methyl (-CH3) and amino (-NH2) groups. The unbranched, hydrophilic, discrete-length molecules have the form Methyl-PEGn-Amine, where the subscript 'n' denotes 4, 8, 12, or 24 ethylene glycol units. The terminal primary amine of each compound provides a specific target for crosslinking and other conjugation methods, making these compounds useful as PEGylation reagents.

Applications of PEGylation:
• PEGylate carboxylate or amine surfaces
• Add inert mass to proteins, immunogens, drug compounds and probes
• Improve solubility (decrease aggregation) of proteins or peptides without affecting function
• Protect proteins from proteolysis

Methods of Covalent Attachment:
• Conjugate to carboxyl groups using EDC and Sulfo-NHS
• Crosslink to primary amines using DSS, BS(PEG)5/BS(PEG)9 or other NHS-ester reagent
• Crosslink to sulfhydryl groups using Sulfo-SMCC or SM(PEG)n reagent
• Attach to oxidized carbohydrate groups (aldehydes) by reductive amination

Why PEGylate a protein or peptide?
Methyl-capped 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, also called polyethylene oxide (PEO), 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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MA(PEG)8 Methyl-PEG-Amine Compound
MA(PEG)12 Methyl-PEG-Amine Compound
MA(PEG)24 Methyl-PEG-Amine Compound

Pierce Plant Total Protein Extraction Kit Thermo Scientific™

The Thermo Scientific Pierce Plant Total Protein Extraction Kit effectively extracts protein from all kinds of dry and fresh plant tissue without liquid nitrogen or organic solvents.

Features of the Plant Total Protein Extraction Kit:
Efficient—obtain excellent protein yields in less than 10 minutes using a single buffer and a spin column
Versatile—native or denaturing lysis buffers for protein extraction from leaves, stems, and seeds
Compatible—extracts can be quantified using the BCA Protein Assay Kit or the Rapid Gold BCA Protein Assay Kit

The Pierce Plant Total Protein Extraction Kit is composed of optimized buffers and filter cartridges that allow for efficient and rapid protein extraction in less than 10 minutes. The kit is designed to rapidly extract denatured or native proteins from plant tissues (leaves, seeds, soft stem, roots, etc.). Simply grind the sample in the lysis buffer in the filter cartridge and centrifuge. The protein extracts can be used for applications such as SDS-PAGE, western blotting, immunoprecipitation, affinity purification, and activity assays.

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Halt Protease & Phosphatase Inhibitor Cocktail, EDTA-free (100X)
Pierce Rapid Gold BCA Protein Assay Kit
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Guanidine-HCl Thermo Scientific™

Thermo Scientific Pierce Guanidine-HCl can be easily dissolved and pH-adjusted to any concentration.

Features of Guanidine-HCl:

• No wasting valuable research time weighing and dissolving guanidine crystals
• Free from UV absorbing materials in the range of 225-300nm
• Free from heavy metal contaminants
• Free from degradation products such as ammonia
• Excellent stability

Applications of Guanidine Hydrochloride:
• Solubilizing proteins from inclusion bodies
• Increasing solubility of hydrophobic peptides and proteins
• Denaturing proteins

Chaotropes, substances that disrupt the structure of water interactions, help to solubilize hydrophobic proteins and peptides for a variety of biological purposes. For example, hydrophobic peptides and proteins may be dissolved in coupling buffer containing either 6M guanidine-HCl or 4M urea prior to immobilization on AminoLink Plus Support to create an affinity column. However, because the degradation products of guanidine and urea include ammonia, which contains a primary amine that will compete for reaction to the support, urea and guanidine solutions must be prepared with high quality reagents immediately before use.

Guanidine is a general protein denaturant, unfolding proteins and altering their three-dimensional structure. Consequently, some proteins will be irreversibly altered upon interaction with guanidine solutions and may lose their binding function. Before any large-scale use of guanidine, it is best to test a small sample and determine whether the denaturing effects will adversely affect the intended use of the protein.

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MS(PEG)4 Methyl-PEG-NHS-Ester Reagent Thermo Scientific™

Thermo Scientific Pierce MS(PEG)n reagents are methyl-terminated, polyethylene glycol compounds (n equals 4 to 24 PEG units) activated as NHS esters for covalent pegylation of primary amines on proteins (e.g., lysines) or assay surfaces.

Features of MS(PEG)4:

• NHS-activated for efficient PEGylation of primary amines at pH 7-9; reaction of NHS-ester group results in formation of stable, irreversible amide bonds
• Fully characterized PEGylation reagents with defined PEG chain lengths; molecules of discrete molecular weight for consistency of performance in protein-modification applications
• Provided as a series of 4, 8, 12 and 24 ethylene glycol units, enabling modification procedures to be optimized for a specific application while retaining all the benefits associated with protein PEGylation
• PEG spacer provides unique advantages, including increased stability, reduced tendency toward aggregation and reduced immunogenicity
• Easy-to-follow instructions increase the likelihood of a successful outcome

MS(PEG)n is the abbreviation for a set of compounds having polyethylene glycol (PEG) spacers with methyl (—CH3) and amine-reactive NHS-ester groups at opposite ends. The unbranched, hydrophilic, discrete-length molecules have the form Methyl-PEGn-NHS Ester, where the subscript 'n' denotes 4, 8, 12, or 24 ethylene glycol units. The N-hydroxysuccinimide (NHS) ester is spontaneously reactive with primary amines (—NH2), providing for efficient PEGylation of proteins, peptides and other amine-containing molecules or surfaces.

PEGylation Applications:
• PEGylate amine surfaces
• Add inert mass to proteins, immunogens, drug compounds and probes
• Improve solubility (decrease aggregation) of proteins or peptides without affecting function
• Protect proteins from proteolysis

Why PEGylate a protein or peptide?
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 biological applications, 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, also called polyethylene oxide (PEO), has these effects because it is nontoxic, nonimmunogenic, hydrophilic, water soluble and highly flexible.

Advantages of Discrete-length mPEG-NHS Ester Compounds
These reagents are specially synthesized as homogeneous compounds of discrete chain length and defined molecular weight. As such, they enable precise control and optimization of surface protein modification experiments. By contrast, typical preparations of PEG compounds are heterogeneous mixtures composed of multiple chain lengths and a range of molecular weights.

Related Products
MS(PEG)8 Methyl-PEG-NHS-Ester Reagent
MS(PEG)12 Methyl-PEG-NHS-Ester Reagent
MS(PEG)24 Methyl-PEG-NHS-Ester Reagent

ML(PEG)4 Methyl-PEG-Lipoamide Compound Thermo Scientific™

Thermo Scientific Pierce ML(PEG)4, or methyl-PEG4-lipoamide, is a methyl- and bidentate thiol-terminated compound that contains a 4-unit polyethylene glycol (PEG) spacer and is used to modify quantum dots, silver and gold surfaces and magnetic particles.

Features of ML(PEG)4:

Metal-binding—terminal bidentate thiol reacts spontaneously with silver, gold and other metal surfaces to form strong dative bonds
Methyl—terminal methyl group is unreactive, the primary function of this compound being to coat quantum dot and other surfaces to reduce nonspecific binding in assays
Polyethylene glycol—PEG groups are flexible, non-immunogenic, hydrophilic, and significantly reduce nonspecific binding of surfaces for protein methods
Spacer arm—four-unit PEG spacer makes compound nearly 24 angstroms long

ML(PEG)4 is a bidentate thiol-terminated and carboxylic acid pegylation reagent. The PEG compound has a defined molecular weight and spacer length and is useful for modifying surfaces such quantum dots, self-assembled monolayers and magnetic particles. Functionalization of solid surfaces with polyethylene glycol spacers such as this one significantly reduces nonspecific protein binding.

The use of ML(PEG)4 with CL(PEG)12 in surface modification can form a hydrophilic 'lawn' of methyl ether-terminated PEGs with periodic exposed carboxy -containing PEGs. The exposed carboxylic acid groups can be coupled to affinity ligands using the carbodiimide coupling reaction with EDC and sulfo-NHS.

Typical PEG reagents contain heterogeneous mixtures of different PEG chain lengths; however, our PEG reagents are homogenous compounds of defined molecular weight and spacer length, providing precision in optimizing modification applications.

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MT(PEG)4 Methyl-PEG-Thiol Compound
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