Intended for the CuAAC reaction with proteins, protein labeling buffer protects biomolecules from being damaged by reactive
oxygen species. This reaction can be used for proteins with alkyne or azide groups, as well as cells
or components of cell lysates metabolically labeled with azide groups.
Azide is conjugated
to terminal alkyne, resulting in a five-membered heterocycle (1,2,3-triazole). Both groups (azide and
alkyne) are extremely rarely found in natural biomolecules, so the reaction is highly specific and
effective to handle various tasks.
The reaction proceeds in the
presence of copper (I) compounds and almost does not depend on pH. Optimized for operations with proteins,
protein labeling buffer contains a salt of copper (II) (a stable precursor of catalytically-active
copper (I) compounds), triethylammonium acetate pH 6.8, water-soluble THPTA ligand, and aminoguanidine. It is recommended
to use a freshly prepared solution of ascorbic
acid to reduce copper (II). THPTA ligand in protein labeling buffer speeds up the reaction by stabilizing
catalytically-active compounds of copper (I). The presence of THPTA ligand also allows protein labeling in water
medium (without organic solvents) and, owing to stabilization of copper (I) oxidation degree, minimizes
production of reactive oxygen species (RAS) and prevents them from damaging proteins by oxidizing
histidine, methionine and cysteine. Aminoguanidine in protein labeling buffer prevents chemically reactive
aldehydes (dehydroascorbate hydrolysis products) from binding to side chains of arginine, N-terminal
cysteine, and lysine.
For this reaction, you will need alkyne- or azide-modified protein in azide
sodium-free buffer, dye azide or alkyne, 1.5х protein labeling buffer, and ascorbic acid. It is recommended to perform
steps 6 to 9 under an inert gas (nitrogen or argon).
We recommend the
following protocol for conjugation of modified proteins with dye derivatives:
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An alkyne derivative of bright red-emitting dye ROX (Rhodamine X, Rhodamine 101) for copper-catalyzed click chemistry bioconjugation. Pure 6-isomer.
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Alkyne derivative of water-soluble sulfo-Cyanine3.5 dye for copper-catalyzed click chemistry.
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A water-soluble dye alkyne for copper-catalyzed click chemistry. The fluorophore sulfo-Cyanine3 is a bright and photostable dye for the Cy3® channel.
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Alkyne functionalized, water soluble far red/near infrared emitting fluorophore sulfo-Cyanine5.5.
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sulfo-Cyanine5 is a water-soluble Cyanine5 dye alkyne for click chemistry.
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sulfo-Cyanine7.5 is a near-infrared fluorophore similar to Indocyanine Green (ICG) but possesses higher fluorescence quantum yield. This is an alkyne for copper-catalyzed click chemistry.
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A sulfonated near-infrared dye alkyne for copper-catalyzed cycloaddition with azides.
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Alkyne derivative of TAMRA dye, pure 5-isomer for сopper-catalyzed сlick chemistry.
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Terminal alkyne for copper-catalyzed click chemistry. Tetramethylrhodamine (TAMRA) is a rhodamine dye which is popular in qPCR and other applications. TAMRA is a also a FRET acceptor for FAM.
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