Conjugation of Proteins to NHS-Activated Gold Nanoparticles

Introduction

Compared to carboxylated nanoparticles that require activation with EDC/NHS prior to conjugation, Cytodiagnostics NHS-activated gold nanoparticles, silver nanoparticles, and gold nanourchins are all available and 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 Gold Nanoparticles or NHS-Activated Gold NanoUrchins
  • Protein Resuspension Buffer (supplied with particles above)
  • Reaction Buffer (supplied with particles above)
  • Quenching Solution (supplied with particles above)
  • 10% (w/v) Bovine Serum Albumin (BSA)
  • Conjugate storage buffer: 20mM Tris (pH 8.0), 150mM NaCl supplemented with 1% (w/v) BSA (or other suitable buffer for your protein)

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 gold 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. Add 10 ul of 10% BSA to the vial.
  8. Using a microcentrifuge, centrifuge the vial for 30 minutes using the appropriate speed for the gold nanoparticle size you are using according to table II below.
  9. Remove supernatant containing unbound protein.
  10. Add 100 ul of conjugate storage buffer to the vial to re-suspend your conjugate. 
  11. Repeat step 8-10
  12. Record the UV-VIS spectra of the conjugate using a spectrophotometer, and dilute to desired optical density using conjugate storage buffer.
  13. Store your protein conjugate at 4°C until use.
  14. (Optional Step) Test for successful conjugation using our Conjugation QC Lateral Flow Dipstick Kit.

* Note: Do not re-suspend lyophilized NHS-activated nanoparticles in buffer prior to addition of 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 Kits MIDI Kits
Reaction Buffer 84 ul 840 ul
Diluted Protein Solution 24 ul 240 ul
Total Reaction Volume 108 ul 1080 ul

 

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

 Gold Nanoparticle Diameter Centrifugation Force MWCO Spin Column
5nm N/A Required
10nm 22,000 x g ** Highly recommended
15nm 17,000 x g  Recommended
20nm 10,000 x g Optional
30nm 2,500 x g  Optional
40nm 1,400 x g Optional
50nm 1,100 x g Optional
60nm 900 x g Optional
70nm 700 x g Optional
80nm 600 x g Optional
90nm 500 x g Optional
100nm 400 x g Optional

**For 10nm gold 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 10nm, the use of 100kDa MWCO Spin Columns will still improve product recovery. For centrifugation speeds and time for MWCO Spin Columns, please refer 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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