Conjugation of Proteins to NHS-Activated Silver Nanoparticles

Introduction

Compared to carboxylated nanoparticles that require activation with EDC/NHS prior to conjugation, Cytodiagnostics NHS-activated gold nanoparticles, NHS-activated silver nanoparticles, and NHS-activated gold nanourchins are all shipped pre-activated in a conjugation-ready format. No manipulation of the nanoparticles is required prior to conjugation, which significantly streamlines the workflow, and more importantly, improves overall conjugate quality. As with carboxylated nanoparticles these pre-activated nanoparticles are suitable for conjugation of proteins and other amine containing ligands.

A recommended starting protocol for conjugation can be found below. Note that the amount of protein added may need to be optimized for your particular protein.

Materials

  • NHS-Activated Silver Nanoparticles 
  • Protein Resuspension Buffer (supplied with particles above)
  • Reaction Buffer (supplied with particles above)
  • Quenching Solution (supplied with particles above)
  • Conjugate storage buffer: 20mM Tris (pH 8.0), 150mM NaCl supplemented with 1% (w/v) BSA.

Procedure

  1. Allow all reagents to warm to room temperature before use.
  2. Dilute (or dissolve) your protein/antibody to a final concentration of 0.5 mg/ml using the supplied protein re-suspension buffer. 
  3. In a microcentrifuge tube combine your diluted protein with reaction buffer according to table I below.
  4. Transfer 90 µl of your protein/reaction buffer mix prepared in step 2 to one of the vials containing lyophilized NHS-activated silver nanoparticles and immediately mix well by pipetting up and down*. 
  5. Incubate the vial at room temperature for 2 hours.
  6. Add 10 µl of quencher solution to the vial to stop the reaction.
  7. Using a microcentrifuge, centrifuge the vial for 30 minutes using the appropriate speed for the nanoparticle size you are using according to table II below.
  8. Remove supernatant containing unbound protein.
  9. Add 100 ul of conjugate storage buffer* to the vial to re-suspend your conjugate. 
  10. Record the UV-VIS spectra of the conjugate using a spectrophotometer, and dilute to desired optical density using conjugate storage buffer.
  11. Store your protein conjugate at 4°C until use.

* Note: Do not resuspend lyophilized NHS-activated nanoparticles in buffer prior to addition of your protein. NHS rapidly hydrolyzes in aqueous solution and may result in loss of conjugation efficiency.

Table I. Quantities of each reagent to mix and add to a single vial of lyophilized NHS-activated nanoparticles.

  3 or 10 Reaction NHS Kits MIDI NHS Kits
Reaction Buffer 84 ul 840 ul
Diluted Protein Solution 24 ul 240 ul
Total Volume 108 ul 1080 ul

Table II. Recommended centrifugation speeds for protein conjugated silver nanoparticles. A centrifugation time of 30 minutes is generally sufficient for a 1 ml sample in a 1.5 ml microcentrifuge tube.

 Silver Nanoparticle Diameter  Centrifugation Force (x g) MWCO Spin Column
10nm 21,000 Highly recommended
20nm 17,000 Highly recommended
30nm 11,000 Optional
40nm 3,000 Optional
50nm 2,000 Optional
60nm 900 Optional
80nm 500 Optional
100nm 300 Optional

 *For 10nm silver nanoparticles the recovery is estimated to be approximately 50% at this particular speed. For better recovery, 1) use an ultracentrifuge to achieve higher speeds or 2) use 100kDa MWCO Spin Columns (if molecular weight of the conjugated protein is <100kDa). While not required for sizes above 20nm, the use of 100kDa MWCO Spin Columns will still improve product recovery. For centrifugation speeds and time for MWCO Spin Columns, please refer to Table III below.

Table III. Recommended centrifugation speeds and time for MWCO Spin Columns.

 Spin Column Size
 		Centrifugation Force Centrifugation Time
0.5 mL 10,000 x g 10 min.
4 mL 1,700 x g 10 min.
15 mL 1,700 x g 10 min.

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