Necroptosis is a form of programmed necrosis, in which protein MLKL (mixed lineage kinase-domain like) plays a key role. After the activation MLKL forms oligomers, translocates to plasma membrane and causes the release of cell content. There are also other necroptotic proteins that have important role in this signalling pathway, including kinases RIPK1 and RIPK3. Necroptosis occurs when caspases are inactive and after the stimulation of death receptors, Toll-like receptors or as a result of interferon mediated response. Necroptotic signalling pathway can be inhibited by necrostatins, which are commonly used when studying different diseases associated with this form of cell death. The mechanisms of initiation, regulation and inhibition of necroptosis are therefore complex and proceed through numerous signalling pathways. The intracellular location of protein MLKL and its translocation to plasma membrane can be monitored by the usage of different fluorescent labelling systems. By tagging the key necroptotic protein we could track its position during the process of necroptosis, which would provide an insight into the characteristics of this form of cell death. Different protein tags, which are fused with target protein, are used for labelling intracellular proteins. The simplest are fluorescent proteins, which emit fluorescence after the excitation with specific wavelength. There are numerous tags classified in this group, including green fluorescence protein, its homologs and mutants, flavin-based fluorescent proteins and bilin-binding fluorescent proteins. Fluorogen-activating proteins, which emit fluorescence only after binding to fluorophore, are also frequently used. The binding reaction can be catalysed by different enzymes, which attach the ligand to its own residue or to another protein. This group of peptide fluorescent tags includes systems HaloTag, SNAP-Tag, CLIP-Tag and TMP-Tag.
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