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  • Title: Enhanced detection efficiency of genetically encoded tag allows the visualization of monomeric proteins by electron microscopy.
    Author: Fukunaga Y, Higashihara A, Nishino Y, Yasunaga T, Jin M, Miyazawa A.
    Journal: J Electron Microsc (Tokyo); 2012 Aug; 61(4):229-36. PubMed ID: 22508950.
    Abstract:
    A cadmium-binding, genetically encoded protein tag, consisting of three repeats of metallothionein (3MT), can be used in electron microscopy for the visualization of multimeric- but not monomeric-tagged proteins due to insufficient electron density in monomeric proteins. Here, we present a technique for detecting monomeric 3MT-tagged green fluorescent protein (GFP-3MT) using a platinum compound to intensify the electron density. Substitution of cadmium by platinum as a result of incubating purified cadmium-binding 3MT-tagged GFP (GFP-Cd-3MT) with cis-diamminedichloroplatinum(II) (cisDDP) was assessed by a UV absorption band centered at 284 nm thereby indicating platinum-sulfhydryl bonds. The incubation time and the concentration of cisDDP to reach maximal absorption were 2 h and 36-fold molar equivalent of cisDDP, respectively. GFP-Pt-3MT isolated by gel filtration chromatography contained 29 platinum atoms per single GFP-3MT molecule. Electron-dense particles were observed in a GFP-Pt-3MT sample by electron microscopy without negative staining. Further image processing and image analysis demonstrated that particles with higher density relative to their surroundings were detectable in both GFP-Cd-3MT and GFP-Pt-3MT samples. These results demonstrate that replacement of cadmium with platinum, together with proper image analyses, improve detection efficiency and enable the visualization of 3MT-tagged monomeric protein by electron microscopy.
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