I've done this on isolated virus particles using the same procedures as I would for sections. Have a look at my papers from my contributions. I would expect your project is not any more complicated.

The Tailless Icosahedral Membrane Virus PRD1 Localizes the Proteins Involved in Genome Packaging and Injection at a Unique Vertex and

Actin Associates with the Nucleocapsid Domain of the Human Immunodeficiency Virus Gag Polyprotein

Brent E Gowen I tried the antibody labeling going off of your paper's protocol and somehow the protein fibrils I should have seen were not visible and the gold labeling seemed pretty sporadic.

I tested just the glutaraldehyde fixation and neutralizing step on my protein fibrils, and that seems to be causing problems. I could no longer make out details in the fibrils, everything was sort of "fuzzy", and there was a lot less protein on the grid after all the washes it seems. The glutaraldehyde makes proteins stick to each other, so I don't think it would make the protein adhere to the formvar any stronger.

I am also wondering if the glutaraldehyde could have affected the antibody binding region of the protein aggregates since there was no fibril alignment of the gold nanoparticles.

Yup, glutaraldehyde can chew up isolated small proteins. I once fixed isolated tubulin that formed microtubules and they looked pretty bad after the glutaraldehyde step.

Put the solutions for 1) to 8) on some clean parafilm. Have a water saturated kimwipe nearby and cover everything with the lid of a petri dish during incubations = humid chamber. Place the grid on top of the solutions; they will float on the top of all the drops. Move the grid from solution to solution; 50 uls solution per drop is plenty.

This is all done at room temperature;

1) incubate your fibrils onto the grids until you know many are stuck (as previously tested by negative stain TEM).

2) place on 2 of drops of PBS with 1 % ovalbumin, 5 minutes each drop.

3) incubate with primary diluted in PBS-Oval. I use no longer than 1 hour.

4) wash the grids 3 times in just PBS-Oval, 5-10 minutes each time.

5) incubate with gold conjugated secondary diluted in PBS-Oval for 1 hour.

6) wash the grids 3 times in PBS-Oval, 5-10 minutes each time.

7) wash the grids in a couple of distilled water washes, 1 minute each drop.

8) negative stain the material as normal.

For a control, rather than incubating with primary, have a couple of grid just incubated with PBS-Oval. Eliminate step 3 for a couple of grids.

Attached is an example of the results on some microtubules labeled for tubulin. Notice there is little gold stuck to the carbon coated formvar of the film holding the microtubules.

So you didn't need to fix your sample before labeling? Also I was wondering about setting the grids to float on droplets, especially with the gold secondary, wouldn't the antibodies settle to the bottom rather than associate with the floating grid?

In the paper I first referenced I used glutalaldehyde and triton x. I had to "crack" the virus samples so that some of the antibodies could get inside the virions where their antigen were suspected to be. You don't need to do this for your sample as you just want surface locations of the fibres. Your fibres don't need to be fixed. You don't need triton X in your sample. Sorry about missing that distinction between the my virus and your sample.

Floating the grids on the drops of solutions is a standard procedure. Most people do this. The gold conjugated secondary is too light within the solvent to settle. You'd have to spin it to get the gold down.

Check your secondary stock solution. Is there an obvious settling of gold ? If the stock solution was old and/or contaminated such that the secondaries clump, then I could see settling happen. Otherwise you'll see no settling within the original secondary solution.

Cheers

B

Thank you very much for your help. This may be a silly question, but if my beta amyloid protein readily attaches to the TEM grid, then what is stopping the ovalbumin from attaching to the grid?

Good question. Does ovalbumin stick to the grid/sample ?

It does stick. It functions as a blocking agent to stick to highly charged material on the grids, thereby preventing immunoglobulins from false positively sticking to these materials. As Ovalbumin is a very small molecule (385 amino acids, its relative molecular mass is 42.7 kDa) it doesn't contribute to the image when used at 1% in PBS.

I have been using PBS-Ovalbumin for TEM immunolabeling for 30 years since I learnt the technique from Dr. Moise Bendayan, U Montreal, Canada. His papers from the 70's and 80's explain the immunolabeling process well. In one paper (I can't remember which one) he explains the advantages of using ovalbumin compared to the other blocking agents used for immunolabeling. You should track down some of his papers.

I've used PBS-Ovalbumin for TEM immunolabeling on both isolated samples (example: virus and isolated cellular bits and pieces) and on sections of embedded material (virus, fish, plants, cultured cells etc). I have labeled Epon, LR White, LR Gold, Lowicryl HM20, and MBM sections of embedded material. I can't think that I would have missed ovalbumin on the grids if it was interfering/sticking and staining on the grids. I've just not seen any direct visual effect of using PBS-Ovalbumin.

You've got me worried now !

Cheers

B