I am trying to locate a 250kDa protein complex. The negative stain image of the complex looks fine with decent looking particles of around 20nm diameter. I am however unable to clearly distinguish the particles on the cryo grid. I have attached images for reference.
I am using a C-Flat 300mesh 1.2/1.3 Holey carbon grids. Some of the sample preparation parameters are as follows:
Glow discharge: 15mA, 15s
Blot force: 0
Blot time: 3s
Protein concentration: 2.5 mg/mL
I know it sounds like a statement of the obvious, but negative stain is not cryo. A sample optimised for neg stain will almost certainly not translate to cryo conditions well - particularly concentration! If you didn’t need to dilute your sample for the negative stain, you almost certainly will struggle to find particles in cryo. Try searching around the edges of the holes, your protein might not be present in the centres if ice is too thin. Also, have a look on the carbon; protein will still stick there in cryo - if you can see particles on carbon and not in the holes, there are a lot more options for adjusting grid conditions.
Also, if just checking cryo conditions (i.e. finding particles is troublesome) crank the defocus up a long way - 4-8um defocus - you want to see what you’ve got, and increasing defocus is the quickest way to boost contrast. If you can see particles at higher defocus, then it becomes the usual game of optimising conditions. Once there, collect at more normal defocus ranges.
I did dilute my sample for negative stain. The negative stain was taken at 70ug/mL while for cryo I used 2.5 mg/mL. I can attach an image which I got from the carbon.
Not much visible there. Ice at the hole edge looks like it might have melted, maybe too thin? Try carbon film, and check holes at higher defocus? At least, that would be my next trial.
Agree with everything @rbs_sci has said - if you can’t see them, they are not there, particularly for something of this size. what does an FFT of your initial image look like, and what is the defocus? That will give you some insight into ice thickness (based on the water ring).
I would suggest trying thin carbon/graphene (at lower protein concentration) or trying high-CMC detergent additives (at higher protein concentration).
A sample optimised for neg stain will almost certainly not translate to cryo conditions well - particularly concentration! If you didn’t need to dilute your sample for the negative stain, you almost certainly will struggle to find particles in cryo. Try searching around the edges of the holes, your protein might not be present in the centres if ice is too thin. Also, have a look on the carbon; protein will still stick there in cryo - if you can see particles on carbon and not in the holes, there are a lot more options for adjusting grid conditions.
Once there, collect at more normal defocus ranges. 
