1-866-344-3954

Conjugation of Proteins to NHS-Activated 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 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


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 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 (gold nanoparticles) or table III (silver nanoparticles) 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 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 µl 840 µl
Diluted Protein Solution 24 µl 240 µl
Total Volume 108 µl 1080 µl



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
5nm Use 100kDa MWCO Spin Column
10nm 17,000 x g 
15nm 15,000 x g 
20nm 5,500 x g 
30nm 2,000 x g 
40nm 900 x g 
50nm 600 x g 
60nm 500 x g 
70nm 400 x g 
80nm 400 x g 
90nm 300 x g 
100nm 300 x g 



Table III.
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.

Gold Nanoparticle Diameter  Centrifugation Force
10nm 21,000 x g *
20nm 17,000 x g 
30nm 11,000 x g 
40nm 3,000 x g 
50nm 2,000 x g 
60nm 900 x g 
80nm 500 x g 
100nm 300 x g 

*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.


Related Products