I have this gold nanoparticle - protein complex in solution. I am trying to isolate protein from the gold nanoparticle to retrieve the protein back in solution and centrifuge out the nanoparticle from solution. What would be the best gold nanoparticle binding molecule/surfactant which I can add in excess and which strongly binds with GNP, thus dislocating the protein attached on its surface? I used Glutathione in excess but it did not do the job.
P.S. I check if the protein is isolated from the GNP by running an SDS page gel. If the GNP is still there in sample, the protein sticks to its surface even on denaturing and thus does not form a band in page gel.
Try sds-page with glutathione or cysteamine. Do not boil the sample with the buffer. You could also try with urea and a thiol molecule.
In theory, using smaller and easily diffusing thiol molecules (e.g. beta-mercaptoethanol, b-ME) may be a good competitor to displace the protein from the gold surface. Since, b-ME is a component in the SDS-loading buffer, just increasing its concentration may do the trick.
Maybe, it would be better to run an agarose gel (0.6% w/v) and loading the sample in the middle of the gel. [i.e., Casting the wells in the middle of the gel. Sample mixed with SDS-loading buffer. Boiling of the sample can be skipped as Omar Gonzalez-Ortega mentioned. Agarose gel prepared and run with SDS-buffer. Staining the gel with coomassie-blue staining.] This would allow visualization of the protein bound to the GNPs without the need of displacing them from GNP surface. You could also see many bands separating based on the number of proteins bound per GNP.
Just curious, how many cysteines are there in your protein ?
I have to be contrary to the answers above from my esteemed colleagues.
The Au-S bond is very tight and I would recommend boiling the Au NP with excess b-mercaptoethanol. It will render the Au NP useless, but it will release any proteins bound to the NP through -SH bonds.
Shubaash Anthiya, I am currently running the Agarose gel with wells in middle. But I am planning to run just native gel, as I thought based on your suggestion avoiding SDS would be better. My protein does not have any cysteine, just 3 methionines.
If there are no cysteine residues, could you elaborate on how did you generate the protein-np complex.
Hi Amar,
The sulphur group [HOOC-CH(NH2)-CH2-CH2-S-CH3] from Methionine probably would not involve in strong S-Au interaction as cysteine thiol group [HOOC-CH(NH2)-CH2-SH]. Depending on the pH, different aminoacids can electrostatically interact with the gold surface.
I think SDS is not a problem. SDS will give negative charge to the protein and hence, all the complexes would migrate towards positive electrode.
The idea of running an agarose gel is to visualize large, high-molecular weight GNP-Protein complexes. [Something similar to the results from the following article, Article “Flash” Preparation of Strongly Coupled Metal Nanoparticle C...
].Running a native gel would be interesting to see the original interaction between the protein and GNPs.
Hi Amar, Omar brings up a critical point. If your protein does not have free thiols, it will unlikely bind covalently to Au particles.
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