Data sheet 6-Methyl-Tetrazine-PEG - Jena Bioscience

Data sheet
6-Methyl-Tetrazine-PEG 4
-Maleimide
Cat. No.
CLK-A139-10
CLK-A139-25
CLK-A139-100
Amount
10 mg
25 mg
100 mg
Applications
Protein-protein conjugates
Protein-antibody conjugates
H 3 C
N
N
N
N
N
H
O
O
O
O
O
N
H
O
O
N
O
Peptide-small molecule conjugates
18F radiolabelling
Protein-oligonucleotide conjugates
Molecular Formula: C 29 H 39 N 7 O 8
Molecular Weight: 613.66 g/mol
Purity: > 95 % (HPLC)
Appearance: solid
Solubility: Chloroform, DCM, DMF, DMSO
Storage conditions: store at -20 °C
Shelf life: 12 months
For research use only!
Surface modification
Descriptions
The inverse-electron demand Diels-Alder cycloaddition reaction
of Trans-Cyclooctenes (TCO) with tetrazines is a
bioorthogonal reaction that possesses exceptional kinetics
(k > 800 M -1 s -1 ) and selectivity. Such excellent reaction
rate constants are unparalleled by any other bioorthogonal
reaction pair described to date.
The extremely fast kinetics and selectivity enables the
conjugation of two low abundance biopolymers in an
aqueous and otherwise complex chemical environment
through the formation of a stable dihydropyridazine. This
bioorthogonal reaction possesses extreme selectivity and
biocompatibility, such that the complimentary reagents
can form covalent bonds within richly functionalized
biological systems, in some cases, living organisms. The
TCO-tetrazine click reaction is a very powerful tool in
catalyst-free protein-protein bioconjugation.
Advanced Features
• By far, the fastest kinetics among any other
bioorthogonal reaction pairs
• Reactions complete in 30-60 minutes at low protein
concentrations (5-10 µM)
• Tetrazine functional group remains stable in aqueous
buffered media (weeks at 4°C, pH 7.5)
• Conjugation efficiency > 99 % without requiring a
toxic catalyst (e.g. Cu(I))
Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
http://www.jenabioscience.com
Last update: May 22, 2013

Data sheet
6-Methyl-Tetrazine-PEG 4
-Maleimide
Important Product Information
• Molecules to be reacted with maleimide compounds
must have free (reduced) sulfhydryls. Reduce peptide
disulfide bonds with disulfide reducing reagents
such as TCEP Disulfide Reducing Gel (Pierce Biotechnology).
Reduce disulfide bonds in high molecular
weight proteins using 5 mM TCEP (1:100 dilution)
for 30 minutes at room temperature, followed by
TCEP removal using a desalting column. Proteins
(e.g., antibodies) can be inactivated by complete
reduction of their disulfide bonds. Selective reduction
of hinge-region disulfide bonds in IgG can be
accomplished with 2-Mercaptoethylamine x HCl (2-
MEA). Sulfhydryls can be added to molecules using
N-succinimidyl S-acetylthioacetate (SATA) or 2-
iminothiolane x HCl (Traut’s Reagent), which modify
primary amines.
• Do not use buffers that contain sulfhydryl-containing
components (e.g., DTT) or azides.
• Avoid buffers that contain azides.
• The maleimide group reacts predominantly with free
sulfhydryls at pH 6.5-7.5, forming stable thioether
bonds. At pH values > 7.5, reactivity toward primary
amines and hydrolysis of the maleimide groups
can occur. At pH 7, the maleimide group is 1,000
times more reactive toward a free sulfhydryl than to
an amine.
• Reactions between tetrazine and TCO are complete
in 30-60 minutes at 5-10 µM.
Additional Material Required
• Water-miscible organic solvent such as dimethyl sulfoxide
(DMSO) or dimethyl formamide (DMF)
• Reaction buffer: Phosphate-buffer (100 mM sodium
phosphate, 150 mM NaCl, pH 7.5) or other suitable
amine-free buffer at pH 6.5-7.5. Include 5-10 mM
EDTA to help prevent the reoxidation of disulfides by
trace divalent metals.
• Spin Desalting Colum (e.g. ThermoScientific Zeba )
Procedure for Labeling Proteins
• Buffer exchange proteins into phosphate reaction
buffer at 1-5 mg/ml using a desalt spin column.
• Immediately before use prepare 5-20 mM 6-Methyl-
Tetrazine-PEG 4 -Maleimide reagent in DMSO or DMF.
• Add a 20-fold molar excess maleimide reagent to
the protein sample and incubate for 1 hours at room
temperature or for 2 hours at 4°C.
• Note: The reaction solution may appear cloudy as
a result of the low aqueous solubility of 6-Methyl-
Tetrazine-PEG 4 -Maleimide; usually, such solutions become
clearer as the reaction proceeds. Many proteins
will precipitate when the DMF or DMSO concentration
exceeds 10 % of the final reaction volume;
if protein solubility is not an issue, there is no limit to
the DMF or DMSO concentration that may be used.
• Quench reaction by adding quenching buffer at 10-
50 mM final and incubating for 15 minutes at room
temperature. Alternatively (or in addition) remove
the excess reagent by desalting the labeled protein
through a desalt spin column or by dialysis.
Protein-Protein Tetrazine/TCO Conjugation
• Calculate volume tetrazine-labeled protein (1-5
mg/ml) equivalent to a 2-5 fold molar excess over
desired volume TCO-labeled protein (1-5 mg/ml).
• Mix calculated volume tetrazine-labeled protein with
desired volume of TCO-labeled protein.
• Allow reaction to proceed for 60 minutes at room
temperature.
• Store conjugate at 4°C until ready for purification or
use.
• Quenching buffer: concentrated (0.5-1 M) cysteine,
DDT or other thiol containing reducing agents
Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
http://www.jenabioscience.com
Last update: May 22, 2013

Data sheet
6-Methyl-Tetrazine-PEG 4
-Maleimide
Troubleshooting
Problem: No or poor labeling of protein with Tetrazine
• Possible reason: Maleimide hydrolyzed
- Allow product to equilibrate to room temperature
before opening
• Possible reason: Thiol-contaminants in protein labeling
reaction buffer (e.g. Glycine, Tris)
- Buffer exchange proteins into an amine-free buffer
before labeling (e.g. 100 mM sodium phosphate,
150 mM sodium chloride, pH 7.5)
Selected References:
Devaraj et al. (2009) Fast and Sensitive Pre-Targeted Labeling of
Cancer Cells through a Tetrazine/trans-Cyclooctene Cycloaddition.
Angew. Chem. Int. Ed. 48:7013.
Haun et al. (2009) Probing Intracellular Biomarkers and Mediators of
Cell Activation Using Nanosensor and Bioorthogonal Chemistry. ACS
Nano. 5:3204.
Blackman et al. (2008) Tetrazine Ligation: Fast Bioconjugation Based
on Inverse-Electron-Demand Diels-Alder Reactivity. J. Am. Chem. Soc.
130:13518.
Devaraj et al. (2008) Tetrazine-Based Cycloadditions: Application to
Pretargeted Live Cell Imaging. Bioconjugate Chem. 19:2297.
• Possible reason: Sub-optimal reaction conditions.
- Optimize labeling conditions by altering molar excess
Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
http://www.jenabioscience.com
Last update: May 22, 2013