I would like to preferentially stain for polyvinylpyrrolidone or a globular protein (in a TEM sample) when they are both coating a silver nanoparticle. The common staining methods don't distinguish between the two and using biotinated proteins for gold labeling is not an option. Any suggestions?
Hi, Teodora,
Can you give us a little more information about how you are preparing your samples? Are you drying nanoparticles down on carbon-coated formvar grids, or what is your sample preparation technique?
Yes, exactly, I am letting a drop of particle suspension dry on a carbon-coated grid.
Dear Teodora
If an antibody is available against this protein, you could use the Protein A-gold technique to label the protein.
Best regards,
Dieter
Without sounding trite why does it matter? ie what are you trying to determine and why? It is intriguing that you have both mixed up yet are dying them down.
What 'common' staining methods did you try?
Are you trying to observe the coating or a difference in Ag?
Proteins are often stickier (negative FCD) so a positive heavy metal ion might work.
Tannic acid might help.
How big is the globular protein and the PVP? high defocus might work.
Also there could be a noticeable difference where they are aggregated as the PVP will act as a better spacer than protein (I'd guess).
good luck
Hi, Teodora,
I would assume that you already attempted to stain your protein with aqueous uranyl acetate, say 8% for 10 minutes, while the nanoparticles were still in solution, prior to depositing them on the grid, and that this technique did not work. You may be able to produce electron density in your protein with osmium tetroxide, 1% at room temperature for 1 hour, while your particles are still in solution. This may give you the added advantage of hardening your particles to help them maintain their shape better when you dry them down on your grids, helping to prevent collapse and maintaining a more round appearance. You may need to play around with the time of osmication to vary contrast. In using either uranyl acetate prestain or osmium tetroxide, rinse your particles well before depositing them on your grids. Osmium is very hazardous. Use a fume hood and gloves when working with it and dispose of it properly. If you use nickel grids, it is possible to osmium stain your sample on the grids and then rinse with distilled water after the staining. You could, of course, uranyl acetate stain on the grids, also. Osmium and UA will not react with each other, so you could use osmium, rinse and then use UA, sequentially, as well.
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