I have been prepared bovine serum albumin (BSA) capped gold nanoclusters and it has red luminescent in UV light (the procedure was already established). During TEM analysis (200 kv), nanocluster has been charging and could not identify the particles at higher magnifications. please suggest the ways the sample preparation for TEM measurement or any special conditions need to be for protein analysis in TEM?
TEM output images are of two types including bright field and dark field, with changing this two mode you can get better images.
Protein having lower contrast in compare to metallic Nps.The protein become degraded and destroyed at higher beam energy (200KV). For visualization of protein with gold cluster under TEM,You should try negative staining for enhancing the contrast of your sample with 2% PTA or Uranyl acetate.The electron beam energy should be 120KV or lower.
Hello Shankar,
You can use negative contrast with uranyl acetate 5% (30 min) after grid preparation.
Thanks Oliveira. I have few clarification about preparing the sample, i.e first nanocluster one drop on the copper grid, then will add a drop uranyl acetate or mixing both nanocluster and uranyl acetate and then take a drop from the mixing on the copper grid. which will be better mixing or alone on copper grid during TEM measurement?
Its depend on availability in your lab.but UA or PTA will be better.Just use 2% and air dry for 30min.don't use 5%,its become very strong for protein. Negative staining should be better for applying on the grid after loading the sample.mixing process is not good.it can make clusters and Crystal of salt.
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