Hi all,
Kindly, I am looking for a SEM/TEM`s protocol used for measuring size of self-assembled peptides (nano-structure)
Hi Safaa,
In order to preserve your delicate peptide structure from being destroyed by the electron beam, you need to protect your sample. One of the simplest method is to apply the Negative Staining. Attached please find files with the negative staining protocols.
Vitaly
Hi Safaa, we published a book chapter review some time ago that could help you!
https://www.researchgate.net/publication/236661469_Microscopy_tools_for_investigating_nano-to-mesoscale_peptide_assemblies?ev=prf_pub
Chapter Microscopy tools for investigating nano-to-mesoscale peptide...
Thank you so much Sergio Kogikoski jr! I appreciate your precious feedback!
What does the self assembled peptide look like macroscopically? Is it in solution or a powder of some sort?
Dear Hitesh Changela, If I got your question, the peptide is in solution (organic/water) is going to take one of the secondary structures (alpha helix, Beta sheet, coil-coil)..etc.
Forming nanostructures (nanospheres, rod-like , tubular, tape like...etc)
Usually we use this very simple protocol for TEM visualization of proteins, viruses and molecular constructions:
1. Drop a droplet of solution to Parafilm surface
2. Put to this drop a conventional TEM grid with carbon coated Formvar film
3. After 30 sec detach the grid from droplet and dry it by filter paper on edge
4. Put the grid to droplet of 1% water solution of uranyl acetate
5. After 10 sec detach the grid from droplet and dry it by filter paper
6. Examine the grid by TEM :)
Good Luck!
Denis Korneev! Many thanks for your answer!
My peptides conc. around 1mg/1ml, is this ok for SEM/TEM?
Safaa Kader, may be the concentration is so high and you have some big aggregates instead a monolayer - it is a typical problem in microscopy of proteins. Dilute 1:10 by buffer before the samples preparation.
I am not sure about SEM. Do you have the sufficient resolution for your objects? A typical protein molecule is not more than 10 nm...
Denis Korneev! Thanks again for your reply!
I am using the liquid-phase method for my peptides, my final concentration is around this. I have got nano-particles around 100-250 nm with DLS.
I have tried using buffers but I have got a micro particles?
The particles with 100-250 nm size are aggregates of a lot peptide molecules. Probably, they will be destroyed by drying on the film surface. The size of aggregates is on depend on ionic strength of solution, pH, concentration and electrochemical properties of peptides. If the concentration is so high you have not monolayer (from molecules or aggregates) on the film surface and an examination of this sample is difficult or impossible.
Denis Korneev! Thanks for sharing me these info,
I have dissolved 1mg peptide in 100ul DMF, then dropped wise DI-H2O (0.5-1 ml) to it.
I guess the conc is around 10-20%! I have got nanoparticles within 150-200 nm.
I have tried to get rid of the solvent (SEM/TEM) by dialysis or vacuum drying but things got worse (500-800nm)
Vinod Kumar! regarding fixation with gluteraldehyde, do I need to dry my peptides from solvent before subjecting them to gluteraldehyde?
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