Adsorption of Proteins to Gold Nanoparticles

Background

Gold nanoparticle conjugates have been used for a wide range of biological applications including the use as probes in light and electron microscopy, SPR, Lateral Flow and Aggregation assays . Stable gold nanoparticle conjugates can readily be prepared by passive adsorption due to electrostatic and hydrophobic interactions between the protein and the surface layer of the colloidal gold. A process that generally is optimally achieved at a pH close to the pI of the protein to be conjugated.

An important parameter to also consider when preparing gold nanoparticle conjugates is the amount of protein bound to the gold colloid. If too little protein is adsorbed to the gold surface, aggregation occurs upon addition of electrolytes present in standard buffers. A titration is therefore performed to determine at which protein concentration saturation and colloidal stability is reached.

**Below is a general protocol designed as a starting point for optimization of protein adsorption. Further adjustments are necessary to maximize loading and stability of proteins on gold nanoparticles. Please Contact Technical Support for detailed support. Consider our comprehensive series of Passive Gold Conjugation Kits.

Materials

  • Standard gold nanoparticles
  • 10% (w/v) NaCl
  • 10% (w/v) Bovine Serum Albumin (Millipore Sigma Cat. #)
  • Purified protein to be conjugated (5 mg/ml)
  • 0.5X phosphate-buffered saline (PBS)
  • 0.1M sodium phosphate buffers with pH ranging from pH 6.5-pH 7.8
  • 0.1M borate buffers with pH ranging from pH 8.0-pH 9.4
  • Conjugate resuspension buffer - 1XPBS, 1% BSA, 20% glycerol (optional)
  • UV-VIS spectrophotometer

Procedure

Titration Procedure (Optimization of pH and Amount of Protein)

  1. Transfer 0.5ml of gold nanoparticle solution to a microcentrifuge tube.
  2. Adjust to desired pH by adding 20ul of either 0.1M sodium phosphate or 0.1M borate buffer of a specific pH.
  3. Transfer 10ul of protein solution (0-5mg/ml, diluted in 0.5X PBS) to a separate microcentrifuge tube. 
  4. With a pipette rapidly add the pH-adjusted gold nanoparticle solution to the vial with protein. 
  5. Incubate for 10 minutes at room temperature
  6. Add 0.5ml of 10% NaCl to the vial
  7. Incubate for 5-10 minutes at room temperature. 

  8. Samples with suboptimal pH and protein quantities will rapidly aggregate upon addition of NaCl which can be observed by a colour change of the solution (from red to purple/blue). No colour change will occur in samples with optimal conjugation conditions.

Preparation of Gold Conjugate (Scale-Up Reaction)

  1. Transfer amount of gold nanoparticles needed for your application from the stock to a new tube.
  2. Add protein amount as determined above plus an additional 10%.
  3. Incubate for 30 minutes at room temperature while stirring.
  4. Centrifuge the solution for 30 minutes at the appropriate speed for the gold nanoparticle size used. For more information on appropriate centrifugation speeds of gold nanoparticles of different sizes, see Gold Nanoparticle Handling and Storage.
  5. Resuspend the pellet in PBS supplemented with 0.1% BSA or 1% PEG.
  6. Store at 4°C until use

References

Thobhani, S., Atree, S., Boyd, R., Kumarswami, N., Noble, J., Szymanski, M., Porter, R.A. (2010) - Bioconjugation and characterization of gold colloid-labelled proteins Journal of Immunological Methods 356, 60-69

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