Aladdin’s DBCO Protein Labeling Kit (100 µg scale) (D1510875) provides a convenient method to label proteins with dibenzylcyclooctyne (DBCO) for use in strain‑promoted azide‑alkyne cycloaddition (SPAAC) reactions (Figure 1). The kit uses DBCO-PEG5-NHS ester, a heterobifunctional crosslinker that readily reacts with primary amines, forming covalently attached DBCO groups that can be further reacted with an azide‑labeled coupling partner to produce diverse bioconjugates. The kit includes reagents for DBCO‑labeling and purification of ten 100μL samples with a concentration of 0.5–5 mg/mL. Labeling and purification can be performed in as little as 90 minutes. The kit is optimized for labeling proteins with molecular weights between 20 kDa and 150 kDa.
Figure 1 Schematic of SPAAC
Aladdin’s DBCO Protein Labeling Kit features include:
- Versatile—label proteins (20–150 kDa) with volumes of 0.5–5 mL
- Chemoselective—DBCO reacts selectively with azides
- Heterobifunctional—reactive towards amine and azide
- Stability—The PEG chain enhances water solubility and reduces non-specific adsorption
- Convenient—labeling and purification can be completed in as little as 90 minutes with 30 min hands-on time
Required materials not supplied
1. Variable‑speed benchtop microcentrifuge
2. 0.5mL microcentrifuge tubes
3. Desired Protein for labeling (free of BSA or any carrier protein)
4. PBS buffer (pH 7.2-7.4).
5. UV‑Vis spectrophotometer
Matters needing attention
1. The purified proteins should be in a buffer that does not contain primary amines (for example, ammonium ions, Tris, glycine, ethanolamine, triethylamine, glutathione) or imidazole. All of these substances significantly inhibit protein labeling.
2. Impure proteins or proteins stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.
3. The crosslinker is moisture‑sensitive. Equilibrate the vial to room temperature before opening to prevent moisture condensation inside the vial. After preparing the stock solution with the anhydrous solvent provided in the kit, it can be aliquoted and stored at –20 °C, where it remains stable for two months. Storage at –80 °C allows for even longer preservation.
4. The sample loading volume for Spin Desalting Columns must be between 70 and 120 μL to ensure optimal performance. Loading volumes outside this range may lead to lower protein recovery or inefficient removal of unconjugated reagents.
5. Do not reuse Spin Desalting Columns.
Instructions for Use
1. Prepare the protein sample
1.1 Prepare the target protein in a buffer (pH 7-9) free of amines, ammonium ions, or azide as these will interfere with the labeling reaction. If necessary, dialyze or exchange the target protein into an appropriate buffer, such as PBS (1X), before labeling.
1.2 Prepare the protein sample at a concentration of 0.5–5 mg/mL in PBS solution. The final volume should be ≤120 μL.
2. Calculations
The optimal amount of DBCO-PEG5-NHS will depend on the desired degree of labeling. In general, higher protein concentrations or proteins with a greater number of accessible primary amines (e.g., lysine residues) on the surface will require less DBCO-PEG5-NHS to achieve the desired labeling density. For recommendations on DBCO:protein ratios, see Table 1.
| Protein concentration Range | Molar Ratio (DBCO:Protein) |
| 0.5-1.0 mg/mL | 40:1-20:1 |
| 1.0-5.0 mg/Ml | 20:1-10:1 |
Table 1. Recommended molar ratio of DBCO-PEG5-NHS to target protein
2.1 Use the following formula to calculate the amount (in millimoles) of crosslinker to add to the sample for an X:1 molar ratio.
2.2 Use the following formula to calculate the volume (in μL) of 20 mM crosslinker to add to the sample.
2.3 For 100 μL of 2 mg/mL IgG (MW = 150,000 g/mol) with 20:1 molar ratio, add 1.3 μL of 20 mM DBCO-NHS to the prepared sample.
3. Protein labeling procedure
3.1 Equilibrate DBCO-PEG5-NHS ester to room temperature.
3.2 Just before use, reconstitute the crosslinker. Add 215 μL of DMSO to the vial of DBCO-PEG5-NHS to obtain a 20 mM stock solution. Vortex or pipette up and down until the solution is homogeneous.
3.3 Add the calculated volume of 20 mM DBCO-PEG5-NHS to the tube.
3.4 Incubate the reaction mixture for 1 hour at room temperature or 2 hours on ice.
4. Prepare the spin column
4.1 Loosen the cap on a spin column, twist the tab off of the bottom, then place the column into a collection tube.
4.2 Centrifuge the column‑tube assembly at 1,000 x g for 2 minutes to remove the storage solution.
Note: When using a fixed-angle rotor, place a mark on the side of the column that faces away from the rotor center. For all subsequent centrifugation steps, place the column in the microcentrifuge with the mark facing away from the rotor center.
4.3 Discard the flowthrough, then place the column back into the collection tube.
4.4 Add 500 μL of PBS Buffer, then centrifuge the column‑tube assembly at 1,000 x g for 2 minutes to equilibrate the column. Discard the flow through.
4.5 Repeat step 4.4 3 times, discarding the buffer from the collection tube each time.
5. Purify the DBCO‑labeled protein
5.1 Transfer the equilibrated column into a new collection tube.
5.2 Carefully pipette the entire reaction mixture (≤120 μL) onto the center of the column.
5.3 Centrifuge the column‑tube assembly at 1,000 × g for 2 minutes. The purified DBCO‑labeled protein is in the collection tube.
6. Determine the Degree of Labeling (optional)
The efficiency of the conjugation reaction can be determined by measuring the absorbance of the protein at 280 nm and the absorbance of the DBCO group at its excitation maximum (309 nm).
6.1 For samples with high concentrations, dilute a small amount of the purified conjugate with PBS.
6.2 Measure the absorbance of the protein at 280 nm (A280) and the DBCO group at 309 nm (A309).
6.3 Calculate the concentration of the protein in the sample using the following formula.
Where:
1. εprotein: Molar extinction coefficient of protein at 280 nm. Generally, the molar extinction coefficient of IgG at 280 nm is 203,000 cm⁻¹M⁻¹, and this also applies to IgA, IgD and IgE.
2. 0.90 is a correction factor for the DBCO contribution to A280.
6.4 Calculate the degree of labeling (DOL) using the following formula.
Where:
1. εDBCO: Molar extinction coefficient of the DBCO group at 309 nm is 12,000 M⁻¹cm⁻¹
7. Conjugate the DBCO‑labeled protein to an azide (optional)
7.1 Prepare the azide‑labeled coupling partner (supplied separately; Cat. No. A1510878; A1510880; A1510881) in an azide‑free buffer, such as PBS.
7.2 Add the prepared azide‑labeled coupling partner to the DCBO‑labeled protein at a 1.5 to 10 fold molar excess.
7.3 Incubate the reaction mixture for 4–12 hours at room temperature.
7.4 Depending on your application, you can use the conjugated protein immediately or purify the conjugated protein by size exclusion chromatography columns and resins.
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