Thiols bind more strongly to gold than histidine (hard-soft acid-base theory. Depending up on the size of the gold one can attached several thiols (~1 thiol per 2-3 gold atom); pH should be kept slightly acidic to prevent disulfide formation.

Do you need one thiol per gold particle? 

Thank You for the useful information! Yes, I need one thiol per gold nanoparticle which is for sure the challenge. Is there any idea to adjust the pH regarding the isoelectric point of the protein?

Actually, one performs a "titration."

First pI of your protein.

Bring gold sol above the pI of the protein.

Add serially diluted protein

Add NaCl to make final conc. 1 M in NaCl.

Check all the solutions; those with low protein concentration salt out gold or solution looks purple - borderline stability. One can scan UV-vis spectra of gold is >2 nm; will show typical  absorption band at 520 nm. 

Choose one conc. above the stable salt . Add BSA (forgot exact conc.) or commercial stabizymes.

If one need gold for electron microscopic studies then they can be pelleted and resuspended by centrifugation. For uniformly sized gold one needs to perform gradient ultra-centrifugation.

For details, please check: Colloidal Gold: Principles, Methods, and Applications by Roth.

Use of colloidal gold in diagnostic surgical pathology by Michael J. Warhol

Thank You very much. I will test your suggestions asap.

You could try a maleimide derivative of InnovaCoat gold. This material already has a protective surface coat so you won't need the passivation step. The coating later is almost impossible to remove and thus provides a very stable support for covalent attachment of the thiol to the maleimide surface. The thiol-maleimide bond is far more secure than a direct thiol-gold bond which can easily dissociate, as you say, during passivation with PEG-SH.  

Do you expect the gold-thiol binding too weak to allow for stable coupling? Since I my aim is the coupling of one protein to two AuNPs, a strong dissociation of gold.thiol would be quite problematic.

Gold-thiol binding is reasonably strong but you can still displace molecules from gold surfaces if you add a competing thiolated molecule. (I think you were seeing this issue with your PEG-SH blocking agent?). Depending on the application the reversible nature of the Au-S bond may be seen as a positive thing, in other cases perhaps it would not. If you need strong binding of protein to the surface, you can attach your thiolated molecule to the InnovaCoat gold-maleimide derivative. The binding will be through the thiol, your histidine tags are of no significance in this situation. You will not break the thiol-maleimide bond simply by adding another thiol. However, you will not need to add a blocking thiol (PEG SH) in this case as the entire gold surface is pre-blocked by the coating layer before you attach your thiolated protein. i.e your protein does not actually bind to the gold metal, it binds to the maleimide groups in the coating layer.

If you want to attach your thiolated protein directly to gold metal you will have to use a blocker that has relatively low affinity for gold, so as not to displace your thiolated protein.

 Dr. Nick Gee: 

What is the thickness of the protective layer of InnovaCoat gold? Is the thickness same for all sizes of gold?

I am planning to do fluorescence quenching/enhancement experiment with quantum dots.

Commercial quantum dots of all sizes apparently are stabilized by a coating of same thickness.  

Also, will stabilizing agent used for InnovaCoat gold interfere in my quenching/enhancement experiments?

Thank you.