We have spotted our lipopeptide sample on glow discharged carbon coated copper grid (1 min) and used freshly prepared 2 % uranyl acetate (2 min). But TEM images are not coming well. Please advise.
I saw you images. What kind of buffer are you using? Sometime high glycerol or detergent buffer creates problem. How do you make your staining solution? Do you adjust the PH properly? You can try 2 % Uranyl formate.
I have not seen your images. What type of imaging issues are you having?
I have found it is best to let your UA sit overnight before using it. Did you filter it? Using a centrifuge also helps to minimize UA precipitation. I've always used just 1% UA in water for negative staining.
How thick is the carbon membrane? Are these commercial grids or did you make them?
Are you seeing salt precip. artifacts from your buffer?
How long prior to imaging your grids did you make them? The longer they sit around, the more hydrocarbons build up and cause contamination. Either a brief glow discharge or UV/Ozone treatment after drop casting can help to minimize issues.
Are you drop casting or spin coating your sample on the grid?
Finally, have you considered other staining agents such as PTA?
Dear Rituparna, You'll need to take into consideration the phase diagram of the lipopeptide .. Apologies if that sounds like teaching you to suck eggs. pH and amphiphile concentration will be important in the phase diagram. Below the critical micellar concentration you should be able to see individual molecules by low angle rotary shadowing. Above the critical micellar conentration you will presumably get spherical micelles. Go higher in concentration and other interesting phases might appear ! Negative staining with Uranyl acetate is quite complicated as the slow uranyl ions will have an effect on the phase diagram and the amphiphile conentration will increase as the stain+ amphiphile dries. Do you have enough material for polarised light microscopy. That's where I would start with trying to understand how this interesting protein behaves.