Conjugation of Azide-Tagged Targets to DBCO-Functionalized Gold Nanoparticles | Nanopartz™
To conjugate azide-tagged antibodies with DBCO-functionalized gold nanoparticles (AuNPs), you can use the copper-free click chemistry reaction between the azide and the DBCO (dibenzocyclooctyne) groups. This reaction is highly specific, biocompatible, and efficient. Here’s a step-by-step guide to functionalizing azide-tagged antibodies to DBCO-functionalized gold nanoparticles:
Materials Needed
- DBCO-functionalized gold nanoparticles: Ensure they are well-dispersed in a buffer compatible with the azide click chemistry.
- Azide-tagged antibodies: Purified and appropriately diluted for conjugation.
- Buffer solution: A compatible, non-reactive buffer such as PBS (phosphate-buffered saline) with a neutral pH (7.0-7.4) is typically suitable.
- Optional blocking agents: BSA or PEG, which may be useful for stabilizing the functionalized nanoparticles post-conjugation.
Functionalizing gold nanoparticles using the azide-DBCO reaction.
Step-by-Step Conjugation Process
1. Prepare Your Solutions
- Dilute the DBCO-functionalized gold nanoparticles in PBS to a suitable working concentration (e.g., 10-20 nM, depending on nanoparticle size and desired loading).
- Prepare a solution of azide-tagged antibodies at an appropriate concentration for the reaction. This will depend on the intended antibody-to-nanoparticle ratio, typically starting with a molar ratio of 100:1 to 10:1 (target).
2. Mix Antibodies with Nanoparticles
- Add the azide-tagged antibodies directly to the DBCO-functionalized gold nanoparticles solution.
- Gently mix the solution to ensure even distribution but avoid excessive agitation, which can destabilize the nanoparticles.
3. Incubate for Conjugation
- Incubate the mixture at room temperature for 12-24 hours, allowing the DBCO-azide click reaction to occur.
- The incubation time may vary depending on concentration and reaction conditions but usually is complete within a few hours at room temperature. The reaction can also be done at 4°C overnight if needed for antibody stability.
4. Purify the Conjugated Nanoparticles
- Following incubation, remove any unconjugated antibodies via centrifugation (if possible without disrupting your AuNPs) or by using a size-exclusion column or ultrafiltration.
- Wash the pellet (if centrifuged) with PBS to remove residual unreacted materials, ensuring the solution is free from non-bound antibodies.
5. Stabilize and Store
- Resuspend the conjugated nanoparticles in a suitable storage buffer (often PBS with BSA or PEG as stabilizing agents).
- Store the conjugates at 4°C, protected from light to preserve both the antibody and nanoparticle stability.
Tips for Success
- Concentration Optimization: Adjust the antibody concentration and incubation times as needed based on preliminary testing to maximize conjugation efficiency.
- Stability Check: DBCO-Azide conjugates are generally stable, but handling and storage in a light-protected environment is beneficial.
- Sterility: For in vivo applications, ensure sterile conditions during the preparation process.
This protocol allows for efficient, site-specific conjugation of antibodies to gold nanoparticles, which is ideal for targeted delivery, imaging, and diagnostics. Let me know if you need more specifics for optimization or troubleshooting.
For more information and to explore their products, visit the Nanopartz™ website. Happy researching!
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