PROTEIN MODIFYERS

See also: Labeling reagents - Cross-linkers - Buffers

1. Selection guide to protein modification tools

2. Quantitation of chemical groups (SH, NH2, biotin)

3. Chemical cleavage

4. Chemical Conversion of Chemical groups

5. FeBABE (3D structure study)

Selection guide to protein modification tools

Target

Cat.#

Product

Use

-S-S-

UP28425

DTT (Cleland‘s reagent)

TS*

MSDS

Cleaves disulfides bridges, introduces sulfhydryls

-S-S-

UP24221

TCEP

MSDS

Alternative to DTT, works in a broader pH range, very selective

-SH

UP01566

DTNB

-

Sensitive and quantitation of amines

-NH2

UP02727

OPA

-

Detection and quantitation of amines

-immob. NH2

UP35300

SulfoSDTB

-

Detection of immobilized amines

-NH2

UP84235

SATA

-

Converts amines to protected sulfhydryls

-NH2

UP42425

2-Iminothiolane (Traut’s reagent)

-

Converts amines to sulfhydryls

-NH2

UP69380

sNHS-acetate

-

Blocks primary amines

-COOH

UP04594

NHS and sNHS

-

Activates COOH groups, used during EDC conjugations

Try

UP20955

BNPS-Skatole

-

Cleaves Tryptophane residues

Arg

UP36862

HPG

-

Modify Arginine residues

Cys

UP99476

FeBABE

MSDS

Cleaves peptidic bound spatially-close to Cysteine residues

Biotin

UP05361

HABA

-

Quantitation of chemical groups (SH, NH2, biotin)

DTNB(Ellman’s reagent)

UP01566H

1g

Sulfo,SDTB

UP35300A

50mg

UP01566I

5g

A sensitive reagent for the quantitation of sulfhydryls

• Useful for monitoring SH content of peptides and proteins before and after reaction of SH-reactive agents

• Very strong detection is measured at 412nm

Detects immobilized amines

SulfoSuccinimidyl-4-o-(4,4-DimethoxyTrityl)-Butyrate

MW 665.6

5-5’-DiThiobis-(2-NitroBenzoic) acid

MW 396

CAS [69-78-3]

Gaur (1989)

Technical Sheet

Related product

N-Acetyl-L-Cystein, UltraPure, MW163, UP04901V1, 5g

AcCys is used as a sulfhydryl standard for the DTNB method

Technical Sheet

OPA powder, Ultrapure

UP02727A

1g

HABA

UP05361D

1g

Reagent for Amines Detection and Quantitation

OPA is reactive toward amines to form an unstable complexe. The complexe can be detected spectrophotometrically at 560nm, or be fluorescence (exc.@330-390nm, emission@436-475nm).

Uptima offers a ready to use and stable formulation with a complete protocole to quantitate amines, in proteins and peptides, or any other biochemical bearing primary or secondary amines.

Technical Sheet

• Works over a wide range of pH and salt concentration

• Works for both purified and complex samples

The HABA when binding to avidin, produces a yellow-orange colored complex which absorbs at 500 nm. Biotin displaces easily the HABA from the complex, causing the absorbance to decrease. The biotin present in a sample can thus be determined by interpolation from a standard curve of free biotin, then the number of moles of biotin per mole of biotinylated protein can be calculated.

o-Phtalaldehyde

MW 134.1

CAS [643-79-8]

2-Hydroxyazobenzen-4'-Carboxylic Acid

MW 242.24

CAS[1634-82-8]

Related product

N-Acetyl-L-Lysin, UltraPure, MW188, UP09111A, 1g

AcLys is used as an amine standard standard for the OPA method

Very sensitive immunoassay for biotin:

inquire (under development)

Related product

avidin     UP39860

biotin      UP10685

Technical Sheet

Chemical cleavage

Proteins may be cleaved at specific size by chemical reaction. Most useful cleavage sites are :

Cleavage site

Product

Applications / Feature

Cys-Cys

Disulfide bond

DTT

TCEP

Dissociation of polymeric proteins and complexes

Denaturation of proteins

• Before electrophoresis analysis (i.e. SDS-PAGE)

• Before labeling (i.e. with Thiol reactive biotinylation agents)

• Before making a conjugate (i.e. with Crosslinkers)

Cys

-

FeBABE

3D-Structure analysis of protein complexes

Try

Tryptophane

BNPS

Specific amino acid cleavage

DTT(Cleland’s reagent)

UP284250

1g

TCEP

UP242214

1g

UP284255

5g

An excellent reducing reagent of disulfides in proteins

• Replaces advantagely the pungent b-mercaptoethanol

• Ultrapure grade (>99.4% SH content)

DL-Dithiothreitol

MW 154.25

CAS [27565-41-9]

Replaces DTT for most exigent requirements

• Reduces completely and selectively alkyl disulfides in mild conditions: do not react with buried sulfhydryls or other chemical moieties

• Works in a broad range of pH (7-9) and better than DTT at low pH

• water soluble and resistant to air oxidation

• Odorless

Applications / Literature

Reduction of samples before SDS-PAGE

Reduction of dimeric Cys-containing peptides

Zahler (1983)

Related product

SH can be dosed with DTNB #UP01566

Technical Sheet - Security Sheet

Tris(2-carboxyethyl)phosphine hydrochloride

MW 286.65

CAS [51805-45-9]

Solubility > 310g/L

Applications / Literature

Reduction of biomolecules for structure studies, conjugations

Kirley (1989), Rueg (1977), Burns (1991)

Incubate 3µM of TCEP with pure IgG for only 15 min at room temperature

Technical Sheet - Security Sheet

How to reduce a peptide?

Add 10mM DTT to 5mg/ml peptide in phosphate 0.1M pH 6, incubate 30 min, desalt

BNPS-Skatole

UP20955A

1g

HPG

Arginin modification

UP36862

Cleaves tryptophan residues in peptidic chains

2-(2'-Nitrophenylsulfonyl)-3-methyl-3-bromoindolenine

MW 363.2

APG

Arginine specific cross-linking

UP28071

Applications / Literature

Hunziker (1980), Fernandez (1985), Fontana (1972)

Technical Sheet

 Sulfhydryl modification

Cleavage of the vicinity of cys residues. Great for 3D structure analysis of protein complexes!

Chemical Conversion of Chemical groups

2-Iminothiolane 

(Traut's reagent)

UP42425A

500mg

SATA

UP84235A

100mg

1g

Converts amines to sulfhydryls

• Reacts with free SH at pH7-10 in water

• Useful for designing oriented conjugates

Converts amines to protected sulfhydryls

• Prevents disulfide formation (ex.peptides)

• Easy deprotection in mild conditions

2-iminothiolane.HCl

MW 137.6

CAS [4781-83-3]

Applications / Literature

Jue (1978), Ghosh (1990)

Technical Sheet

N-succini-midyl S-acetylthioacetate hydrochloride

MW 231.2

CAS [76931-93-6l]

Applications / Literature

Can also be used fas a crosslinking agent by thiolating biomolecule then exchanging sulfhydryl with an other molecule

Duncan (1983)

Related products

DMSO, HydroxylamineHCl, DTNB #UP01566

Technical Sheet

SulfoNHS-acetate

UP69380A

100mg

HPG

UP36862A

100mg

Blocks primary amines

• Water soluble

• Efficient at pH7-10

Modify arginine residues

• Reacts in mild conditions (pH7-9)

• Arginin specific

• Can be monitored at 340nm

Sulfo-succinimidyl-acetate

MW 259.2

CAS [4781-83-3]

Applications / Literature

Modification of peptide or proteins

Binding site studies

Technical Sheet

• More resistant than p-nitrophenylglyoxal

• More soluble than phenylglyoxal

b-Hydroxyphenyl Glyoxal

MW 168.2

CAS [76931-93-6l]

Applications / Literature

Yamasaki (1980)

Technical Sheet

NHS

Use to activate COOH groups

UP04594

DTT

Converts disulfide bridges to free sulfhydryl

UP28425

Sulfo-NHS

As NHS, but water soluble

UP54422

EDTA

Chelating agent of divalentions

UP03629

Speciality

For the study of ion channels and transport proteins, as well as enzymes and receptors

FeBABE

UP994760

1mg

-

A unique tool to study tridimentional structures!

• Binds to Cys-SH residue then cleaves peptide or DNA chain within 12 A viccinity

• Quick reaction

Bromoacetamide group binds to free sulfhydryls, and introduces an Iron chelate. The chelate cleaves spatially-close DNA or peptidic bond within 10 sec to 20 min in presence of ascorbic acid and H₂O₂.

b-BromoAcetamidoBenzyl-EDTA Iron (III) chelate

Elucidation of 3 dimensionnal structures (E.Coli cytochrome bd quinol oxidase, a subunit of E.Coli polymerase…)

Used too as a chemical nuclease

Rana (1991), Ghaim (1995), Murakami (1995), Miyake (1998), Owens (1998), Deriemer (1981)

Reducing agents DTT #UP28425, TCEP #UP242214

Technical Sheet - Security Sheet

Desalting the excess of reagent? See Dialysis

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