I have this soluble and stable protein which I believe to be in complex with gold nanoparticles of small size. My solution contains gold nanoparticles and these proteins bound on its surface. I want to check if they are bound or not by comparing the band intensity of 10 µL sample, 10 µL control sample containing same concentration of protein but no gold ions/NP, 10µL of supernatant after hard centrifugation (to check for un bound proteins) and 10 µL of precipitate (to check for bound proteins). To check if they are indeed in complex I decided to run SDS PAGE and results were as attached.
I was expecting the columns should add up, but it seems based on B, C and D, I have lost protein somewhere and its not showing up
L: Ladder
A: 1.5 µM protein control
B : protein-gold complex (containing 1.5 µM protein)
C : supernatant after centrifugation (gold nanoparticle- protein complex precipitate seen from color of solution, not shown)
D: precipitate
I boiled each sample in sample buffer and then took the supernatnt of each to make sure Gold nano particles did not enter the gel.
Where did the protein go from the sample?
Hi,
The protein seems to have gone with the gold-nanoparticles.
Since, there is no protein band in the lane B, all the protein must have complexed with the gold nanoparticle. Either the gold-NP+protein complex are too heavy to enter the gel or most of them were left in the precipitate.
I assume that the interaction between protein to gold nano-particles is by thiol-gold (S-Au) bonding. If the boiling did not dissolve the gold nanoparticle (i.e. Au-Au bond is not broken by boiling), then it would not have released the protein from gold-NP surface. Since, Au-S covalent bond has almost same strength as Au-Au bond [1].
[1] Hannu Häkkinen. The gold–sulfur interface at the nanoscale. Nature Chemistry volume 4, pages 443–455 (2012). doi:10.1038/nchem.1352.
You could titrate with standard amount of gold nanoparticles with different concentrations of protein, in small volume, say 10uL. Prepare the samples without centrifugation and load all the sample into the wells to see at which concentration of protein you are able to detect free protein. Since, the gel will separate based on molecular weight, free and nanoparticle-bound proteins will produce separate bands. This would tell you the saturation limit for gold nanoparticles.
Best Wishes.
There are no special methionine's located on the surface of the protein and the protein seems to be encapsulating the gold nanoparticles due to presence of negative charge on proteins surface and (debatable) positive surface charge on gold. This is just an hypothesis. But your suggestion of Au - S interaction seems right as during boiling with sample buffer, the protein will denature and expose its methionine and stick to gold surface tightly, i believe.
I will definitely try your idea of checking the maximum concentration of free protein for a standard GNP solution. Just one question about it, would this standard GNP need not have any capping agent ? and how should I synthesize them ? GNP in my experiment were synthesized IN presence of protein.
Thank you for the answer.
Yes, it is possible that the protein-GNP interacts by electrostatic interaction as you mentioned. Additionally, there could be Au-S interactions, if/ when the free thiols are exposed.
To form the covalent Au-S bond, free thiols (R-SH) are required, specifically deprotonated thiol-ions (R-S-) [1].
Methionine would not be able to make strong Au-S covalent bond as the thiol is methylated. However, methionine can reduce the Au-ions under alkaline condition (pH 12) to form GNP [2]. This can be relevant in this case, as the GNP are synthesized in the presence of the protein.
If the protein has any exposed/easily accessible cysteine residues, it will readily bind the gold nanoparticles via Au-S bond, and act as a capping agent. And the reducing property of free thiols can directly lead to the synthesis of gold nano-particles under mild conditions [3].
For synthesis, you can try two different ways:
1. You can use your standard method that you already used. This synthesis is giving GNP+Protein complex with no free protein, as seen from the gel-lane B. This will act as the standard GNP. So for the titration experiment, additional protein can be added to this standard GNP solution.
Capping agent is necessary to avoid aggregation of Au0 nanoparticles. But, anything can act as capping agent by electrostatic interaction. Here, the protein and the reducing agent.
2. To see if you are able to synthesize the GNP just with your protein, in the absence of other reducing agents. If there are accessible free thiols (cysteine), then it should spontaneously form GNP and cap the GNP via Au-S bond. (please see [3]).
Denaturation causing Au-S?
Yes, it is possible that the boiling step denatures the protein and exposes the cysteine thiol residues.
But the other possibility is that, since the GNP are synthesized in the presence of the protein, the initial Au-ions will have access to free thiols even in the interior of the protein. This SH-Au3+ interaction could reduce the gold to Au0 and act as the nucleus for the GNP synthesis around the nucleus.
Effect of Denaturation and effect of protein concentration on GNP-Protein interaction are discussed in these article [4, 5].
To check is the Au-S bonding requires denaturing, you can run a SDS-PAGE with and without boiling step.
References:
[1] Article The gold-sulfur interface at the nanoscale
.[2] Article Gold-Coated Cobalt Ferrite Nanoparticles via Methionine-Indu...
[3] Article Retention of Enzymatic Activity of ??-Amylase in the Reducti...
[4] Article Conformation aspect in the a-amylase induced agglomeration o...
[5] Article Modulating enzymatic activity in the presence of gold nanoparticles
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