I have a protein-gold nanoparticle complex. As I want to run SDS page on the protein sample, I want to eliminate gold nanoparticles from the sample as it is clogging the gel. Centrifuging at high concentration does not solve the problem, as the protein (even after denaturing) stays on the surface of GNP and precipitates with MBP during centrifugation. So I wanted a suggestion for how to reduce the GNP back to gold ions in presence of protein in the solution, and not breaking its peptide chain.
Thank you.
Dear Amar Thaker you can dilute your sample of protein gold nanoparticle complex thereafter centrifuge at very high speed(>20K) RPM then collect supernatant and concentrate through lyophilization, or you can try low concentration acrylamide gel that will may work.
Could you completely describe what you are doing right now for sample preparation?
Hey Omar,
I am synthesizing gold nanoparticles by reducing gold ions with a good's buffer HEPES. In presence of protein the gold nanoparticles formed are mono dispersed and the results are repeatable. I want to run an SDS page of the centrifuged sample, to see the amount of protein attached to the gold nanoparticles and in the free state in supernatant. But I find that when I take the precipitate and add sample buffer to it and heat it, the protein sticks even strongly to the gold nanoparticles. So want to dissolve it without affecting the peptide chain before running on PAGE.
I meant the details once you have the particle with protein. What do you do? Hope you are not boiling the sample.
Hi Amar,
You can use low percentage acrylamide gel as Arvind Singh Chandel suggested. Alternatively, you can test with low percentage (0.2 - 0.7%) agarose gel [1]. Since your aim is to detect the protein, you can use the regular SDS-loading buffer and protein staining technique on agarose gel.
If you wish to dissolve the gold nanoparticles (GNP):
- 1 - you can use strong iodine tincture (7% iodine + potassium iodide/ sodium iodide in ethanol and water) [2]. You may have to find the volume and time required for complete dissolution of GNP. You can run a gel after 1 hour and overnight incubation to see if you have free protein, which will indicate the dissolution of GNP. If the pH of the iodine solution is not too acidic or basic, it might not hydrolyze the protein.
- 2 - Presence of reducing agent might favor GNP formation from dissolved gold ions. So, you can eliminate any reducing agent (dithiothreitol, DTT /or/ beta-mercaptoethanol) from the SDS loading buffer.
You can manually prepare 4x loading buffer with the following composition: 250mM Tris–HCl (pH 6.8). 8% (w/v) sodium dodecyl sulfate (SDS). 0.2% (w/v) bromophenol blue and 40% (v/v) glycerol [3].
References:
[1] C Hasenoehrl et al., (2012) Enhanced detection of gold nanoparticles in agarose gel electrophoresis, Electrophoresis 33(8): 1251–1254, DOI: 10.1002/elps.201100556.
[2] Cody's Lab (Youtube Channel), Precious Metal Refining & Recovery, Episode 4: Recovering gold from RAM, https://youtu.be/HhuwO8AjM7k. Iodine dissolving gold nanoparticles: 12:09 - 12:52. For detailed explanation, see the full video.
[3] SDS-PAGE Sample Loading Buffer (4×); Cold Spring Harbor Laboratory. http://cshprotocols.cshlp.org/content/2015/12/pdb.rec087791.full?text_only=true
Best Wishes.
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