BioReady 80 nm Bare Gold Passive Conjugation Protocol
The following procedure demonstrates the process of pH titration to conjugate an IgG antibody to 250 µL of OD 20 BioReady™ 80 nm Bare Gold. A pH range at 7, 8, and 9 will be evaluated to determine the optimal point for passive binding. If desired, 0.5 or 0.2 (or any other increment) of pH point can also be investigated. A larger range of pH from 6–10 can also be evaluated if necessary with the appropriate buffers (see Further Optimization).
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Since the isoelectric point will differ for each protein used, the optimal pH for passive conjugation should be determined empirically.
It’s important to note that this protocol provides a general guideline only. Optimal conjugation procedures are protein-dependent and optimization techniques will differ for each protein/assay.
Materials Needed
- NanoComposix BioReady™ 80 nm Bare Gold
- Protein buffer exchange/desalting columns
- Protein purification buffer (e.g. 5–10 mM potassium phosphate, pH 7.4)
- 100 mM buffers for titration with pH range 7–9
- ≥ 1 mg/mL protein
- 10% sodium chloride solution
- Conjugate block buffer (e.g. 10 mM potassium phosphate or borate, 10% BSA, pH 7.5–8.5)
- Conjugate diluent (e.g. 10 mM potassium phosphate or borate, 1% BSA, pH 7.5–8.5)
- 1.5–2 mL centrifuge tubes
- Vortexer, rotator, centrifuge, and bath sonicator
Protein Preparation
The protein for conjugation can be purified into a buffer free of additional proteins or salt components. Some commercial products, antibodies for example, may contain additives for stabilization (e.g. BSA) or preservatives (e.g. sodium azide) that need to be removed to increase the efficiency of passive adsorption. Protein can be purified into the appropriate buffer using spin columns or dialysis tubing with the appropriate molecular weight cut-off. We recommend using purified protein for conjugation at a concentration ≥ 1 mg/mL. Refer to the data sheet(s) provided by the supplier for proper storage and handling.
Conjugation Loading
For passive adsorption to 80 nm gold, a typical loading range can be anywhere from 10–120 µg of protein per mL of OD 20 gold. However, the optimal loading can also vary outside of this range depending on the protein used and the assay's performance. For information regarding the optimization of the protein loading, refer to the Further Optimization section.
Without knowing the optimal loading for the antibody, a loading of 40 µg Ab per mL of OD 20 gold (or 10 ug Ab per 250 µL of gold) is chosen for this procedure as a starting point to ensure at least sufficient coating for the pH titration process. The optimal loading can be evaluated after a pH point has been selected (see how to optimize the antibody loading in the Further Optimization section).
pH Titration Flow Chart
Protocol
- Prepare a set of 3 eppi tubes. Label each of the tubes 7A, 8A, and 9A to correspond with each pH point.
- Add 12.5 µL of each 100 mM buffer to its respectively labeled tube. The volume of buffer added should be around 1/20 of the total working volume of gold.
- Add 10 µg of antibody into each of the tubes containing the buffer from step #2.
- Add 250 µL of OD 20 gold to each of the tubes from step #3. Vortex and incubate on the rotator for 10 minutes.
- Aliquot 50 µL of 10% NaCl into another separate set of 3 eppi tubes. Label each of the tubes 7B, 8B, and 9B.
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After about 10 minutes of incubation for the "A" tubes (buffer + Ab + gold):
- Pipette 50 µL of the conjugate from each "A" tube into each respective "B" tube containing NaCl.
- Leave the remaining "A" tube volume to incubate for an additional 20 minutes.
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Vortex the NaCl + conjugate mixture in the "B" tubes. Incubate on rotator for about 10 minutes and observe any color change:
- Stable conjugate: the golf maintains its red color
- Unstable Conjugate: the gold becomes purple, gray, clear, or precipitate.
Move forward with the pH condition(s) that produced a stable conjugate.
- After the "A" tubes from step 6b have incubated for a total of 30 minutes, select the tubes that correlated to the stable "B" tubes. Add 1/10 volume of conjugate block buffer so that the final BSA concentration in each "A" tube is around 1%..
- Incubate for 30 minutes on rotator.
- Centrifuge the conjugate at 3600 RCF for 3 minutes.
- Carefully remove the supernatant and resuspend with conjugate diluent buffer to the target OD (e.g. 200 µL final volume for OD 20). Vortex and bath sonicate if needed to fully resuspend the conjugate.
- Perform UV-Vis reading and check for the final OD.
- Store conjugate at 4 °C until use. Do not freeze.
- Functionally test the selected pH condition(s) on the final application (e.g. lateral flow strips).
Further Optimization
Other pH points can be evaluated by preparing the appropriate buffers at the pH of interest for screening. As an option for further optimization, smaller increments around the selected pH can be done to evaluate the optimal pH point. For example, if both pH 7 and pH 8 resulted in stable conjugates and performed well with the functional testing, a 0.3 pH increment can be done to evaluate pH 6.8, 7.1, 7.4, 7.7, 8.0, and 8.3 to further optimize this process. The following buffers can be used for the evaluation:
| Buffer | Buffering Capacity (pH) |
|---|---|
| MES | 5.5-6.7 |
| HEPES/potassium phosphate | 6.8=8 |
| Borate | 8–10 |
| Carbonate-bicarbonate | 9–10 |
Once an optimal pH point has been determined for the conjugation, the protein to gold ratio can be varied to determine the optimal protein/antibody loading. At the previously determined optimal pH, vary the amount of antibody added to the buffer in step #3. We recommend evaluating a range between 20–200 µg of protein loading to find the protein loading that yields a stable conjugate and has the highest performance in the functional assay. For best results, the volume of protein added to the conjugation should not exceed 10% of the total gold volume as this may dilute the final buffer concentration and cause the pH to drift slightly (concentrate the protein to above 1 mg/mL if necessary).
Other parameters that should be optimized for the assay include protein incubation time, blocking time, raw materials, and chemistry formulations (block buffer, conjugate diluent/storage buffer, running buffer, any pad treatment buffers if needed, etc.). If there are substantial changes to the other components of the assay during development, re-optimization of the conjugate may be required.
Frequently Asked Questions
Can I perform the conjugation at a different OD?
Can I use potassium carbonate to do the pH titration?
Can I use potassium carbonate to do the pH titration?
Can I conjugate any type of antibody or protein?
Is there a test to confirm that my conjugates are functional?
What is the shelf life of the nanoparticles?
What is the shelf life of the conjugates?
What other particles are available for conjugation?
