Molecules, which shall react with maleimide compounds, must have free (reduced) sulfhydryls. Reduce peptide disulfide bonds with disulfide reducing reagents such as Immobilized 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-HCl (2-MEA). Sulfhydryls can be added to molecules using N-succinimidyl S-acetylthioacetate (SATA) or 2-iminothiolane-HCl (Traut's Reagent), which modify primary amines.
Do not use buffers that contain sulfhydryl-containing components (e.g. DTT).
Avoid buffers that contain azides, which can react with DBCO.
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.
Do not use DTT, TCEP or β-mercaptoethanol, because they will reduce the azide.
Additional Materials Required:
Water-miscible organic solvent such as dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF)
Reducing reagents such as Immobilized TCEP Disulfide Reducing Gel (Pierce Biotechnology)
Reaction buffer: Phosphate-buffered saline (PBS) or other sulfhydryl-free buffer at pH 6.5 - 7.5. Include 5 - 10 mM EDTA to help prevent the reoxidation of disulfides by trace divalent metals.
(Optional): Quenching buffer: concentrated (0.5 - 1 M) cycteine, DDT or other thiol containing reducing agents
(Optional): Spin Desalting Columns or Slide-A-Lyzer Dialysis Cassettes
Prepare sulfhydryl-containing protein, prepared as described in the Important Product Information section.
Immediately before use, weigh a small quantity of DBCO-Maleimide and dissolve it in dimethylformamide (DMF) or dimethylsulfoxide (DMSO) at a 5 - 20 mM concentration.
Dissolve protein(s) in Conjugation Buffer at 0.1 mM (e.g. 5 mg in 1 ml for a 50 kDa protein).
Add DBCO-Maleimide solution to the dissolved protein(s) at 0.4 mM final concentration (ca. four-fold molar excess for 0.1 mM protein solution).
Note: The reaction solution may appear cloudy as a result of the low aqueous solubility of DBCO-Maleimide; usually, such solutions become clearer as the reaction proceeds. Many proteins will precipitate when the DMF or DMSO concentration exceeds 10 - 15 % 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.
Incubate reaction mixture for 1 hour at room temperature or for 2 hours at 4 °C.
Quench reaction by adding Quenching Solution at 10 - 50 mM final and incubating for 15 minutes at room temperature. Alternatively (or in addition) remove the excess nonreacted reagent by desalting or dialysis.
Copper-free Click Reaction
Prepare the azide-containing sample in reaction buffer.
Add DBCO-protein conjugate to azide-containing sample.
Recommendation: Add 1 mole equivalent of limiting protein to 1.5 - 3.0 mol equivalents of highest abundance protein.
Incubate the reaction at room temperature for 2 - 4 hours. Incubation at 4 °C requires 4 - 12 hours.
The reaction is now ready for purification.
Long-term aqueous Stability of DBCO-labeled Samples
DBCO modified goat IgG (DOL 7) losses about 3 - 5 % of its reactivity toward azides over 4 weeks at 4 °C or -20 °C. For long time storage azide- and thiol-containing buffers should be avoided.
Direct Measurement of DBCO Incorporation
The degree of DBCO incorporation (i.e. the number of DBCO per protein molecule) can be determined from the absorbance scan of the purified conjugate (235 - 400 nm).
Problem: No conjugation of DBCO with azide
Possible reason: One or more sample is not labeled
- Confirm molecules were labeled or repeat activation process
Possible reason: DBCO-Maleimide decomposed
- Allow product to equilibrate to room temperature before opening
- Prepare new solutions in the indicated dry solvents
- Avoid buffers that contain sulfhydryl
Possible reason: Excess reagent not quenched or removed
- Remove non-reacted reagent by dialysis or desalting
Problem: Low conjugation of DBCO and azide
Possible reason: Suboptimal reaction conditions
- Increase incubation time
- Optimize conjugation conditions by altering molar excess
- Perform conjugation reactions at 37 °C
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Zeng et al. (2012). 64Cu Core-Labeled Nanoparticles with High Specific Activity via Metal-Free Click Chemistry. ACS Nano.6 (6):5209.
Arumugam et al. (2011). [18F]Azadibenzocyclooctyne ([18F]ADIBO): A biocompatible radioactive labeling synthon for peptides using catalyst free [3+2] cycloaddition. Bioorg. Med. Chem. Lett.21:6987.
Campbell-Verduyn et al. (2011). Strain-Promoted Copper-Free Click Chemistry for 18F Radiolabeling of Bombesin. Angew. Chem. Int. Ed.50:11117.
Debets et al. (2010) Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition. Chem. Commun.46:97.