Editing Protein (section)

===Cellular localization===

[[File:Localisations02eng.jpg|thumb|right|upright=1.35|Proteins in various [[cellular compartment]]s and structures tagged with [[green fluorescent protein]] (here, white)]]

The study of proteins ''in vivo'' is often concerned with the synthesis and localization of the protein within the cell. Although many intracellular proteins are synthesized in the [[cytoplasm]] and membrane-bound or secreted proteins in the [[endoplasmic reticulum]], the specifics of how proteins are [[protein targeting|targeted]] to specific organelles or cellular structures is often unclear. A useful technique for assessing cellular localization uses genetic engineering to express in a cell a [[fusion protein]] or [[chimera (protein)|chimera]] consisting of the natural protein of interest linked to a "[[reporter gene|reporter]]" such as [[green fluorescent protein]] (GFP).<ref name=Stepanenko2008/> The fused protein's position within the cell can then be cleanly and efficiently visualized using [[microscopy]].<ref name=Yuste2005/>

Other methods for elucidating the cellular location of proteins requires the use of known compartmental markers for regions such as the ER, the Golgi, lysosomes or vacuoles, mitochondria, chloroplasts, plasma membrane, etc. With the use of fluorescently tagged versions of these markers or of antibodies to known markers, it becomes much simpler to identify the localization of a protein of interest. For example, [[indirect immunofluorescence]] will allow for fluorescence colocalization and demonstration of location. Fluorescent dyes are used to label cellular compartments for a similar purpose.<ref name=Margolin2000/>

Other possibilities exist, as well. For example, [[immunohistochemistry]] usually uses an antibody to one or more proteins of interest that are conjugated to enzymes yielding either luminescent or chromogenic signals that can be compared between samples, allowing for localization information.<ref>{{Cite web |last=Hrycaj |first=Steven |date=17 October 2023 |title=Immunohistochemistry: Origins, Tips, and a Look to the Future |url=https://www.the-scientist.com/immunohistochemistry-origins-tips-and-a-look-to-the-future-71439 |access-date=2024-12-22 |website=The Scientist Magazine}}</ref> Another applicable technique is cofractionation in sucrose (or other material) gradients using [[isopycnic centrifugation]].<ref name=Walker2000/> While this technique does not prove colocalization of a compartment of known density and the protein of interest, it indicates an increased likelihood.<ref name=Walker2000/>

Finally, the gold-standard method of cellular localization is [[immunoelectron microscopy]]. This technique uses an antibody to the protein of interest, along with classical electron microscopy techniques. The sample is prepared for normal electron microscopic examination, and then treated with an antibody to the protein of interest that is conjugated to an extremely electro-dense material, usually gold. This allows for the localization of both ultrastructural details as well as the protein of interest.<ref name=Mayhew2008/>

Through another genetic engineering application known as [[site-directed mutagenesis]], researchers can alter the protein sequence and hence its structure, cellular localization, and susceptibility to regulation. This technique even allows the incorporation of unnatural amino acids into proteins, using modified tRNAs,<ref name=Hohsaka2002/> and may allow the rational [[protein design|design]] of new proteins with novel properties.<ref name=Cedrone2000/>