Have you actually tried cryo EM on any of your samples or have you just done negative staining so far ? Your fibrils may be coalescing during the drying process during negative staining. They may be separate enough prior to freezing for cryo EM.

Have you actually tried cryo EM on any of your samples or have you just done negative staining so far ? Your fibrils may be coalescing during the drying process during negative staining. They may be separate enough prior to freezing for cryo EM.

I've done cryo on similar looking things. A few ideas for you:

- PBS is a terrible buffer for using on neg stain. The P reacts with Uranyl Acetate (which assume you've used) and can exacerbate bundling during the air drying.

- Neg stain should be used with a pinch of salt to give you an indicator of what's going on with you sample and how it looks overall, but when you move to cryo things like sticking to carbon, flattening, skewing and adhesions can be dramatically different. I would try some cryo and before you grid up perhaps try an aggressive vortex or even ultra sonication as well as the native sample.

- Quite often in cryo when you look at these bundles, you have the good fortune that they are quite large and so you can find them easily at low mag. If you look toward the ends of the structures, they will often thin out and provide individual fibre repeats there.

Rich

Hi Brent,

I have only done neg. stain so far. I will cryoEM and update the results.

Hi Rich,

Yes, I am using 2% Uranyl Acetate. I have replaced the PBS with water but then the making fibril process was not successful (all aggregates). I have n't tried any other buffers though since PBS gave the the fibrils. But like you said, I will try the cryo EM first.

Thank you both for your answers.

B

Of those who are interested. I have tried to add NaCl into the fibrils sample to the final concentration of 100 mM (gently mixed and left the sample at RT for 1 hour before carrying out EM negative stain). The fibrils seem to break off a little (see picture). I guess I will increase the NaCl to higher.

P.S. I am waiting to get trained on using the cryoEM machine

Good result. However, be aware that too much salt may adversely affect your ability to image the samples by cryo-TEM.

However again, some people freeze their samples in the negative stain itself. A dude by the name of J Robin Harris has some good references on this procedure. Check out his papers. Examples;

Negative Staining and Cryo-negative Staining: Applications in Biology and Medicine

• Jan 2014
• Methods in molecular biology (Clifton, N.J.)

Negative staining and Cryo-negative Staining of Macromolecules and Viruses for TEM

• Feb 2011
• Micron

@Brent E Gowen, Thank you very much for the info.

Hi Binh (Albert) An Nguyen , Have you tried Tris calcium buffer (20 mM Tris, 138 mM NaCl, 2 mM CaCl2, 0.1 % NaN3, pH 8.0)?

Please see: https://www.nature.com/articles/s41467-019-09032-0#Sec9

Haaris Ahsan Safdari Great materials. I will definitely try this for my next sample.

Thank you.