A recommended starting protocol for conjugation can be found below. Note that the amount of ligands added may need to be optimized for particular biomolecule.
1. Allow all reagents to warm to room temperature before use.
2. With the supplied re-suspension buffer, dilute or dissolve sample oligonucleotide/protein to the final concentration suitable for the particular gold nanoparticle size to be conjugated. Suggested protein Concentration: 5 mg/ml; Suggested Oligonuclotide concentration: 250 µM.
Note: a) Maleimide reacts with thiol groups. Depending on the type of protein for conjugation, cleavage of disulfide bonds, or addition of sulfhydryl groups might be necessary prior to conjugation.
3. In a microcentrifuge tube, combine your diluted ligand from Step 2 with reaction buffer. Reaction Buffer: 60µl; Diluted ligand: 48 µl; Total Volume: 108 µl.
4. Transfer 90µl of sample ligand solution prepared in Step 3 to one of the vials containing lyophilized Maleimide Gold Nanoparticles and immediately mix well by pipetting up and down.
5. Incubate the vial at room temperature for 1 hour.
6. Add 10µl of quencher solution* to the vial and incubate for 15 minutes to stop the reaction.
*The quencher is supplied in a lyophilized format and should be reconstituted with 100 ul of ddH2O just prior to use. Any remaining quenching solution should be stored at -20°C.
7. Using a microcentrifuge, centrifuge the vial for 30 minutes using 100kDa MWC Spin Column for the gold nanoparticle size.
Note: b) For effective conjugation, avoid any other molecules containing thiol or contaminating proteins (e.g. BSA), which would compete with the ligand for binding sites. Consider using BSA Removal Kit for Nanoparticle Conjugation (SR-08-01).
8. Discard the supernatant containing unbound ligand.
9. Add 100ul of gold conjugate storage buffer to the vial to re-suspend the conjugate.
Note: A gold conjugate storage buffer is not supplied with the kit. Use a standard biological buffer compatible with the ligand.
A recommended storage buffer for a protein gold conjugate is 20mM Tris (pH 8.0), 150mM NaCl supplemented with 1% (w/v) BSA and 0.025% Tween 20.
A recommended storage buffer for an oligonucleotide gold conjugate is 10mM Sodium Phosphate (pH 7.0), 100mM NaCl.
10. Record the UV-VIS spectra of the conjugate using a spectrophotometer and dilute to desired optical density using a gold conjugate storage buffer.
11. Store the gold conjugate at 4°C until use.
