This strategy complements its nonfluorescent sister know-how BONCAT that permits the tagging of newly synthesized proteins for selective isolation and identification. FUNCAT is based on the introduction of little bio orthogonal, chemically reactive alkyne or azide groups into proteins by means of metabolic labeling with all the noncanonical amino acid analogs azidohomoalanine or homopropargylglycine. Both amino acid analogs are surrogates for methionine and are integrated into nascent proteins when applied to the extracellular medium and taken up with the kinase inhibitors cells. Hence, the metabolic labeling stage is quite much like classical radioisotope labeling and might be combined with or adhere to drug therapy or electrophysiological stimulation. To improve the fraction of replaced methionine, a methionine depletion step prior to AHA or HPG addition is a good idea, and methionine need to be absent in the medium throughout the metabolic labeling response. The incorporated azide or alkyne groups, as nonbiological reactive handles, serve to distinguish newly synthesized proteins from the pre present protein fraction just before metabolic labeling. Following AHA therapy cells are fixed together with a fluorophore is covalently and chemoselectively attached for the launched practical groups by way of click chemistry a copper catalyzed azide alkyne cycloaddition.
STRATEGIC Arranging The fundamental Protocol describes FUNCAT with AHA metabolic labeling of cultured cell lines and primary cells plated on coverslips or glass bottom dishes, visualization of newly synthesized proteins in fixed cells by chemoselective response with a fluorophore alkyne, plk1 and subsequent immunolabeling.
Three alternate protocols are offered within the following sections to describe variations within the protocol when applying FUNCAT to hippocampal slices, to an entire organism larval zebrafish, and also to hippocampal neurons cultured in microfluidic chamber gadgets. The primary and 2nd approaches visualize protein synthesis in tissue with intact circuitries, consequently they can be completely suited to mix them with electrophysiology or, as in the situation of zebrafish larvae, with behavioral reports. The FUNCAT procedure described in Alternate Protocol 3 is meant to allow compartment specified treatment of neurons an technique to research aspects of local protein synthesis or community pharmacological manipulation. Since the technique is compatible with immunohistochemistry, all protocols contain a section describing submit hoc antibody labeling. The Assistance Protocol can provide a information to mix FUNCAT with substantial resolution fluorescence in situ hybridization. This can be of relevance when bridging the gap involving in situ localization of mRNAs, translation, and also the newly translated proteome.