Receptor tyrosine kinases (RTKs) are an important class of transmembrane receptors that mediate some of the most crucial biochemical pathways essential to the growth, differentiation, and survival of a cell and thus, are highly involved in cancers. Due to the complexity of RTKs having biochemically different domains including a transmembrane domain, an intact crystal structure of any of these proteins remain elusive as it is difficult to produce milligram amounts of intact functional RTKs for crystallography studies. A heavily studied RTK is fibroblast growth factor receptor 2 (FGFR2), which plays a key role in fibroblast growth regulation, differentiation, and oncogenesis. Previous studies have focused on expressing FGFR2’s extracellular, transmembrane, and intracellular domains individually. For this protocol, we have focused on the extracellular and transmembrane domains of the FGFR2 protein. The function of the expressed protein is validated by demonstrating its ability to bind heparin and fibroblast growth factor 1 (FGF1). The primary contribution of our protocol is expressing two RTK domains together, including the transmembrane domain, in milligram quantities. Being able to express RTKs to define its crystal structures would enable pharmacologists to design cancer drugs that selectively target active conformations.

Higher-order chromatin organization shaped by epigenetic modifications influence the chromatin environment and subsequently regulate gene expression. Direct visualization of the higher-order chromatin structure at their epigenomic states is of great importance for understanding chromatin compaction and its subsequent effect on gene expression and various cellular processes. With the recent advances in super-resolution microscopy, the higher-order chromatin structure can now be directly visualized in situ down to the scale of ~30 nm. This protocol provides detailed description of super-resolution imaging of higher-order chromatin structure using stochastic optical reconstruction microscopy (STORM). We discussed fluorescence staining methods of DNA and histone proteins and crucial technical factors to obtain high-quality super-resolution images.