Conjugation of Proteins to NHS-Activated Gold Nanoparticles from Cytodiagnostics

By AZoNano Staff Writers

Table of Content

Cytodiagnostics NHS-Activated Gold Nanoparticle Conjugation Kits
Components of NHS-Activated Gold Nanoparticle Conjugation Kit
Key Features and Applications of NHS-activated Gold Nanoparticle Conjugation Kits
Key Characteristics of NHS-Activated Gold Nanoparticles
Protocol for Conjugation
About Cytodiagnostics

Cytodiagnostics NHS-activated Gold Nanoparticle Conjugation Kits

The NHS-activated gold nanoparticle conjugation kits offered by Cytodiagnostics have been designed for high-efficiency, single-step conjugations of proteins and other primary amine-containing ligands to gold nanoparticles with a diameter range of 5-100nm.

The NHS-activated gold nanoparticle conjugation kit, consisting of ready-to-use pre-made mixtures, does not require activation or manipulation of the gold nanoparticles before conjugation, thus eliminating the formation of poor performing conjugates. The protein can be readily mixed with the pre-activated NHS ester gold nanoparticles provided in the kit.

Cytodiagnostics offers the kits in convenient 3 or 10 small-scale reaction formats, enabling many to be conjugated concurrently and ready for use within 2.5hr. These kits are suitable for screening and optimization purposes before scale-up production, which can be carried out with Cytodiagnostics’ NHS-activated gold nanoparticle conjugation MIDI kits.

Components of NHS-activated Gold Nanoparticle Conjugation Kit

The NHS-activated Gold Nanoparticle Conjugation Kit includes the following components:

  • NHS-Activated Gold Nanoparticles (lyophilized)
  • Quencher Solution
  • Reaction Buffer
  • Protein Re-suspension Buffer

All kit components need to be stored at -20°C. NHS-activated gold nanoparticles are stable for at least three months if stored unopened and as specified.

Key Features and Applications of NHS-activated Gold Nanoparticle Conjugation Kits

The NHS-activated Gold Nanoparticle Conjugation Kit yields covalently bound ligand and more stable conjugate through rapid and simple one-step conjugation reaction without pre-activation requirements. Spacers between the conjugated ligand and the gold nanoparticle surface reduce effects on the tertiary protein structure, which can cause poor performing conjugates, a typical issue that can be observed in conjugates prepared by passive adsorption.

The NHS-activated Gold Nanoparticle Conjugation Kit is suitable for producing protein gold conjugates for a myriad of applications, including lateral flow assays, blotting and microscopy and transmission electron microscopy.

Key Characteristics of NHS-Activated Gold Nanoparticles

NHS-activated Gold Nanoparticles are supplied in ready-to-use lyophilized format. The following are the key characteristics of NHS-activated Gold Nanoparticles:

  • Polydispersity Index (PDI): < 0.20
  • Core diameter: 5-100nm (Coefficient of Variance < 12%)
  • Concentration: OD=20 when dissolved to a final volume of 100µl
  • Surface functionality: NHS-ester (spacer between gold surface and NHS-group)
  • Absorbance (λmax): 515-572nm (size dependent)

Protocol for Conjugation

The following are the steps involved in the recommended conjugation protocol:

  • All reagents are allowed to warm to room temperature prior to use.
  • The protein/antibody to be conjugated is then diluted to a final concentration of 0.5mg/ml using the supplied protein re-suspension buffer.
  • The diluted protein is then combined with reaction buffer in a microcentrifuge tube as per the following table:
  3 or 10 Small Scale Reaction Format Kits Midi Kits
Reaction Buffer 84 µl 840 µl
Diluted Protein Solution 24 µl 240 µl
Total Volume 108 µl 1080 µl
  • Then about 90µl of the resulting protein/reaction buffer mix is transferred to one of the vials loaded with lyophilized NHS-activated gold nanoparticles and mixed well by pipetting up and down.
  • The vial is hen incubated at room temperature for 2hr.
  • Now 10µl of quencher solution is added into the vial to stop the reaction.
  • The vial is then centrifuged using a microcentrifuge for 30min at a speed appropriate for the size of the gold nanoparticles, as given in the following table:
Gold Nanoparticle Diameter Centrifugation Force
5nm 100kDa MWC Spin Column
10nm 17,000 x g, 1hr or 100kDa MWC Spin Column
15nm 17,000 x g
20nm 5,500 x g
30nm 4,500 x g
40nm 2,000 x g
50nm 1,500 x g
60nm 1,125 x g
70nm 900 x g
80nm 600 x g
90nm 400 x g
100nm 400 x g
  • Now the supernatant consisting of unbound protein is removed, followed by the addition of 100µl gold conjugate storage buffer to the vial for re-suspending the conjugate.
  • A spectrophotometer is then used to record the UV-VIS spectra of the conjugate, which is then diluted to desired optical density using gold conjugate storage buffer.
  • The resulting protein conjugate needs to be stored at 4°C until use.

The kit does not contain a gold conjugate storage buffer. Hence, it is recommended to use a standard biological buffer that is compatible with proteins to be conjugated. These products are for research and development purposes, not for household, drug, or other purposes.

About Cytodiagnostics

Cytodiagnostics is a biotechnology company based in Burlington, Ontario, Canada. The company focuses on providing and developing nanotechnology derived products and services for the international life science market. Its goal is provide customers with the highest quality products to ensure success in their research and development efforts.

This information has been sourced, reviewed and adapted from materials provided by Cytodiagnostics.

For more information on this source, please visit Cytodiagnostics.

Date Added: Feb 27, 2014 | Updated: Mar 5, 2014
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