Hi Ted,

The aggregation happens by drying on the grid surface. It is a typical problem for proteins, nanoparticles or small viruses by NS sample preparation. There are some ways to solve it:

1. Decrease the concentration;

2. Change the ionic strength of the solution;

3. Modification of the grid surface.

Good luck!

Have you prepared your coated grids using a glow discharge unit to dispense (neutralize) the substrate charge?  I have done this with virus, phage, and nano-spheres

Regards,

Ken

[email protected]

Hi Ted,

The aggregation happens by drying on the grid surface. It is a typical problem for proteins, nanoparticles or small viruses by NS sample preparation. There are some ways to solve it:

1. Decrease the concentration;

2. Change the ionic strength of the solution;

3. Modification of the grid surface.

Good luck!

Before coating or any part of the process, first get the grids ultraclean-- there are traces of oil that create awful artifacts for just about everything. I have boiled them in straight ethanol and blotted dry on filter paper before another immersion in acetone, then air dry in a hood or desiccator.

Also, if they are copper grids ensure they are bright and shiny, not old and corroded on exposure to air; the microscopic pitting from corrosion is hard to work around, even with coating.

In my work with isolated ribosomes and polyribosomes, it was key to place a small droplet of the concentrate (use a tuberculin syringe needle) in the negative stain on the grid on a filter paper, then use a wedge of filter paper to suck it off, then dry. No aggregates, nice spread and contrast.

Thank you all for your suggestions

@ Ken

Yes, I did glow discharge for 1min right before applying samples.

@Denis

By changing the ionic strength, do you have any suggestions on the conditions? I am thinking about adding maybe 10mM NaCl, do you think that is an OK starting point? And do you think adding some reducing agent such as TCEP would help to combat oxidation? (My protein does not have disulfide bond)

For the grid surface, I used CF300-Cu square mesh and glow discharged for 1min right before sample application. Do you think this is sufficient or there are other tricks to help? I have seen people adding poly-L-lysine but but sure if that really helps?

@Vinod

The results surprised me in that the gel filtration polished samples were not even as good as proteins thawed straight out of the freezer... 

@Kristine

We used commercial pre-coated grids (stored in desiccator) and glow discharged them right before experiments. 

Could you kindly give a bit more detail on the ribosome preparation? I am very interested. Thanks

The best conditions for prevention of the aggregation: ionic strength, pH, etc. depend on the properties (isoelectric point, etc.) of your samples. The exact parameters are individually and it is possible to find it empirically. 10 mM NaCl as a start point? Why not.

In my opinion, the discharging and poly-L-lysine coating are not helpful in your case. These methods are suitable for improving of the adhesion of the particles to grid surface: typical problem with EM virus diagnostic - a grid is empty - without any virus particles, after special treatment (discharging, poly-L-lysine, etc.) adhesion will be better and the virions will be stick to surface. If your do not have an "empty" grid and the problem is just aggregation - it is not necessary to improve the adhesion - it is enough.

The aggregation occurs by drying. It is possible to have 2D-quasicrystalls by a concentrated solution. The main goal is prevention of this process - the answer is in conditions of the solution. It is not in grid surface adhesion properties.

Good luck!                

Try the following:

do not put a drop of your protein sample on the surface of a carbon-coated grid; instead, use diffusion of the protein particles to the surface of the carbon-coated grid by floating the grid on a sitting drop of your sample. You might have a chance to "harvest" more individual protein particles because any aggregates - if present in your sample at this time - would arrive close to the grid surface much later than individual protein particles - vertical diffusion upwards! Washing, blotting dry and negative staining as usual.

Could anyone explain the effect of the glow discharge treatment (in low vacuum - residual air) of the carbon-coated formvar grids ? There are nitrogen and oxygen ions bearing positive and negative charges impinged to the topmost atomic surface layer of the carbon thin film ? In other words: glow discharge produces surface charging or neutralization ? 

Hi Ted,

Since your grids are precoated (ugh!), try dipping them in poly-L-lysine solution (ref tissue culture lit, promotes cell adhesion) then drying, should help both with charge problems and the protein aggregation concentration problem.

Gosh, details on ribosomes was decades ago. As I remember, taken directly from the ultracentrifuged pellet: custom-coated grids were sitting on filter paper; microdroplet applied to grid(s) and most suctioned off with the tip of a filter paper wedge. Air dried under a petri dish to prevent fine particulate contamination.

    Suctioning off most of the droplet was found necessary in order to see whether we were looking at singlet ribosomes or polysomes. See whether procedure is written in any old papers by me and B. G. Atkinson; but so long ago, maybe they're no longer available.

   I suspect the success was due to the suction step! Spread them out nicely.  Good luck-- think like a ribosome.

Valentin,

Glow discharge produces a surface charge on the carbon film. Typically a negative charge is applied to the grid to make the surface hydrophilic, which greatly aids the evenness that particles and stain spread across the grid, the effect is quite dramatic. In some cases a positive charge can be applied and various gasses used instead of air.

Not mine, but from somewhere on the Researchgate. All solutions are the critical thinking about the problem. Proteins in all environments - perhaps not in ice(?) - tend to crystal formation(?). Put a drop on a grid, and one can expect little seeds landing first. Put the grid on the drop, and one should expect - with more time - expect the suspended proteins near the surface of the drop to be unaggregated. One could also experiment with 2x or 10x dilutions and waiting overnight for the surface of each to --- yada, yada. Since this was caim from my memory, that may be gone in August, 2019 (80th). I thought I'd better get this in before I spent looking for the origin, and I beg his pardon for not remember the name.

Dear @ Frederick Monson,

good to see your reply...just as a support for your young 'memory' I 'd like to wish all the best in 2019 ...

and - regarding the interesting question - I would like to add a possible list of publications (as listed in RG's archive) regarding the matter "before I spent looking for the origin..." , try:

https://www.researchgate.net/search.Search.html?type=publication&query=crystal%20%20%20formation%20%20aggregation%20%20%20suspended%20%20proteins%20%20grid%20%20EM%20negative%20stain

and perhaps find the publication you thought of or find -anyway -publications dealing with "negative staining", suspensions, aggregation, proteins and crystal formation....

Best wishes again, W.H.M.