Hi
The protein that I try to conjugate with AuNPs has no cysteine, so I modified my AuNPs with COOH-PEG-SH groups. But the conjugation doesn`t proceed yet. I tried all recommended procedure but I didn`t get desirable results yet.
There is some probability for this observation:
1- the protein I work with may be deactivated after conjugation.
2- the carboxyl group of AuNPs may not been activated using EDC/NHS, however I try all recommended procedures including, MES buffer and deionized water in pH range between 5- 6.5
My question is:
May SH-PEG doesn`t have enough carboxyl group, because I ordered it from a laboratory to synthesize and attach it to AuNPs that I`ve synthesized before. How I can be sure that my PEGylated AuNPs are carboxyl terminated?
My second question is how I can be sure that the protein -AuNPs conjugation without using dot blot method? (My protein is recombinant , and its antibody is unavailable). Is SDS-PAGE applicable?
Dear Samaneh Mirsian thanks for sharing this very interesting technical question with the RG community. There are various helpful literature references about this topic available. For example, please have a look at the following relevant paper:
Conjugation of Polymer-Coated Gold Nanoparticles with Antibodies—Synthesis and Characterization
Article Conjugation of Polymer-Coated Gold Nanoparticles with Antibo...
The good thing about this paper is that it is freely available as public full text on RG, so that you can download it as pdf file. please note that this is just one typical article in this field. You can find and access many more by searching the "Publications" section of RG.
It might also be helpful reading the answers given to some closely related questions which have been asked earlier on RG. For example, please check the following threads:
Conjugation of PEG to AuNPs?
https://www.researchgate.net/post/Conjugation-of-PEG-to-AuNPs
(14 answers)
and
What are the techniques to conjugate antibodies to PEGylated gold nanoparticles?
https://www.researchgate.net/post/What-are-the-techniques-to-conjugate-antibodies-to-PEGylated-gold-nanoparticles
(4 answers)
Good luck with your work and best wishes, Frank Edelmann
EDC/NHS sucks, even when using them in large excess; hydrolysis is the problem. Reductive amination gives better results, but you have to use sh-peg-nh2.
You could thiolate your protein as a way of avoiding edc chemistry and concerns over the quality of your think ligand. Presumably it is possible to check the biological activity of the protein pre and post thiolation? If so, you can then proceed to conjugation with some confidence. To prove that your protein is on the gold surface, you should expect some stabilization of the colloid ie. resistance to salt-induced aggregation. There are far more sophisticated methods too e.g. electron microscopy, but most people would be satisfied with a salt stress test.
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