[3dem] protocols Biomolecule-Gold Nanoparticle Conjugates

Chaix, Denis denis.chaix at Vanderbilt.Edu
Fri Jul 9 13:50:46 PDT 2010


Thank you very much I have already think to use this kind of beads, but the idea is more make a mono layer of peptide around the beads.
I plan to use this protocol and I will see if that works, 
Attachment of Peptides Directly to Gold Nanoparticles

"""Thiols form covalent bonds to metals such as gold and silver. Thiol-gold chemistry is being employed in a wide range of biomolecule-sensing strategies, including a gold particle colorimetric assay for DNA (10). One of the more appealing aspects of thiol-gold chemistry is that the reaction proceeds at room temperature in aqueous solution. Moreover, attaching many different functional thiols to a single particle is typically as simple as stirring all the ingredients together in one pot (11).

For a 20-nm colloid, the thiolated species are typically added so as to achieve a molar ratio of approx 2500 thiols per nanoparticle. To ensure monolayer coverage, this ratio can be adjusted according to the core size of the colloids; the available number of adsorption sites is directly proportional to the total surface area and thus to the square of the particle diameter. If a mixed thiol monolayer is sought, the desired thiols may simply be stirred in solution together with gold particles and allowed to adsorb. In many cases, the mole fraction of thiols bound to nanoparticles will reflect the mole fraction added to the reaction mixture (11). Mixed monolayers of PEG and peptides are synthesized as follows. Note that all glassware must be clean.
1.  	Combine 72 µL of thiolated PEG (20 µm) and 72 µL of peptide (20 µm) to produce a molar ratio of PEG to peptide of 1:1.
2.  	Add 145 µL of this mixture to 1 mL of 20-nm-diameter citrate-capped gold nanoparticles (1.16 nM) to produce a molar ratio of thiol to gold of 2500:1.
3.  	Stir the mixture of gold/PEG/peptide at room temperature for 1 h to allow complete exchange of thiol with citrate on the gold nanoparticles.
4.  	To purify the complex, centrifuge at 12,000g for 30 min.
5.  	Decant the supernatant and resuspend the pellet in 1 mL of water."""""
in Assembly and Characterization of Biomolecule-Gold Nanoparticle Conjugates and Their Use in Intracellular Imaging
By: Alexander Tkachenko2, Huan Xie2, Stefan Franzen2, Daniel L. Feldheim2

thanks you
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Chaix, Denis
Vanderbilt University
Email: denis.chaix at Vanderbilt.Edu
Dept. Cell and Developmental Biology
________________________________________
From: David Carlson [dbcarlson13 at gmail.com]
Sent: Friday, July 09, 2010 3:36 PM
To: Chaix, Denis
Subject: Re: [3dem] protocols Biomolecule-Gold Nanoparticle Conjugates

Hi Denis,

if your peptide has a 6-his tag and is stable around pH 7.5 - 8.0 you can use 5nm Ni-NTA nanogold from nanoprobes.  I have had a lot of good results binding my protein in 5 times molar excess to my his tag.  Some problems do arise because one gold can bind many proteins, and if your peptide is a dimer it can bind multiple gold particles, but you can purify them away with a sizing column.

This is also true for the 1.8nm Ni-nta gold as well.  However, they are much more difficult to see... best seen in 3-d reconstructions of proteins or the use of STEM.

Hope this is helpful,

David Carlson (4th year graduate student)
Scholey lab, UC Davis
Evans lab, UC Davis

On Fri, Jul 9, 2010 at 1:24 PM, Chaix, Denis <denis.chaix at vanderbilt.edu<mailto:denis.chaix at vanderbilt.edu>> wrote:
Dear All member of this useful mailing list
I want to conjugate peptide (17 kDa) on 5 nm gold beads and I search a protocol to do this.

I found a protocol Assembly and Characterization of Biomolecule-Gold Nanoparticle Conjugates and Their Use in Intracellular Imaging
By: Alexander Tkachenko2, Huan Xie2, Stefan Franzen2, Daniel L. Feldheim2 but I search precisions on storage and characterisation of peptide-gold beads conjugates?
Have you an Idea

thank you



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Chaix, Denis phd
Vanderbilt University
Email: denis.chaix at Vanderbilt.Edu
Dept. Cell and Developmental Biology_______________________________________________
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