Preparation of Disease-Related Protein Assemblies for Single Particle Electron Microscopy
Electron microscopy (EM) is a rapidly growing area of structural biology that permits us to decode biological assemblies at the nanoscale. To examine biological materials for single particle EM analysis, purified assemblies must be obtained using biochemical separation techniques. Here, we describe effective methodologies for isolating histidine (his)-tagged protein assemblies from the nucleus of disease-relevant cell lines. We further demonstrate how isolated assemblies are visualized using single particle EM techniques and provide representative results for each step in the process.
Key wordsRecombinant proteins Histidine tag Fibrocystin Electron microscopy Single particle analysis
This work was supported by NIH/NCI grant R01CA193578 to D.F.K.
- 7.Guay-Woodford LM (2014) Autosomal recessive polycystic kidney disease: the prototype of the hepato-renal fibrocystic diseases. J Pediatr Genet 2:89–101Google Scholar
- 8.Rauchman MI, Nigam SK, Delpire E, Gullans SR (1993) An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse. Am J Phys 3(Pt 2):F416–F424Google Scholar