I don't think that serum proteins are attaching to my nanoparticles when I incubate them in a culture medium with serum during in-vitro studies. I want to isolate the proteins attaching to the nanoparticles to establish that protein adsorption increases the stability of nanoparticles. How can I isolate the proteins adsorbed on the nanoparticles?
You can probably isolate the nanoparticles by a filtration/dialysis that lets small, unbound proteins through, but does not let the NP's through (are np's significantly larger than proteins?).
If you can't use dialysis, you can also centrifuge down the paritcles. NP's are heavy and will spin down but lighter, unbound proteins require very huge forces to be spun down in a solution. The best way to do this is to take a tube of a semi-viscous medium (say 6% glycerin in h20) and carefully pipetter your np-protien solution as an overlayer onto the solution. It should just sit on top without mixnig. Then when you centrifuge, you can be highly confident that excess protein could not get through the semi-viscous medium, while your particles do. You will have to play with various settings, as the optimal settins vary between batches of NP's and their surface coatings.
Let me ask you this: what are you going to do when you isolate them? If you just have one protein in the medium, you can verify that binding occurs perhaps just be looking at the UVVis spectrum before the addition of the protein and after (assuming this doesn't cause particle aggregation).
Thanks so much for the reply. Yes I think adding a semi-viscous solution would help in collecting the heavier nanoparticles from the lighter protein, however I doubt whether the protein attached to the NPs, would also be removed in the process. Anyway I will definitely give it a try.
I was thinking of separating the proteins attached to the NPs by gel electrophoresis and then doing silver-staining. the purpose of my experiment is just to find out if serum proteins are binding/ absorbing to my NP surface.
It's unlikely that the viscous solution will detach the particles unless they are very loosely bound. Yes, you can try the electrophoresis, and as I said, UVVis is also an easy way to measure this.
Good luck.
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes