This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We are using clonable tags (metal precipitating peptides and Metallothionein) in our effort to identify, localize and study proteins in the EM. These clonable tags will play a role similar to what GFP plays in light microscopy. The tags we are currently working with are A3 (a gold precipitating peptide), Ge8 (a silver precipitating peptide) and Metallothionein, MTH (a heavy metal binding protein). Proteins well studied in our lab were used as a vehicle for our experiments. Eg5 (a kinesin motor domain), Kar3 (a kinesin motor domain), Vimentin (an intermediate filament protein), Desmin (another intermediate filament protein) and VP1 (a major capsid protein of the polyomavirus) Constructs made include: Eg5+A3, Eg5+Ge8, Eg5+3A3, Eg5+2MTHs Kar3+A3, Kar3+2MTHs Vimentin+A3, Vimentin+2MTHs, Vimentin+1MTH Desmin+1MTH VP1+A3, VP1+Ge8, VP1+3A3 In our efforts to optimize the metal precipitation and binding reactions by the peptide labels, we conducted a series of trial-and error experiments. These experiments involved preparing sample by a variety of methods, using various concentrations and types of gold precursors, different concentrations of proteins and also different concentrations and types of reducing buffers. After a large number of experiments, we are concentrating our efforts on constructs Eg5+A3, Vimentin+A3, Kar3+A3, Vimentin+A3, Desmin+1MTH and Vimentin+1mMTH which based on our preliminary data show the most promise.
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