蛋白質標記系統啟動試劑盒提供了標記CLIP-tag融合蛋白的所有必需組份。能在活細胞、固定細胞及體外將紅色或綠色熒光基團共價連接到目標蛋白上。每種啟動試劑盒包括一個編碼所選 tag 的質粒和兩種細胞通透性的熒光標記物。試劑盒中還提供一種陽性對照質粒，其編碼有亞細胞定位明確的標簽蛋白（如：膜蛋白、核蛋白等）。如果必要，試劑盒還會提供一個與目的標簽相互作用的、非熒光的陰性對照阻斷劑。The SNAP-tag? and CLIP-tag? are novel tools for the specific, covalent attachment of virtually any molecule to a protein of interest, providing simplicity and extraordinary versatility to the imaging of proteins in live and fixed cells, and to the study of proteins in vitro. The creation of a single gene construct yields a tagged fusion protein capable of forming a covalent linkage to a variety of functional groups, including fluorophores, biotin, or beads. This system provides a powerful and unique tool to study the role of proteins in a variety of highly dynamic processes, including protein trafficking, turnover and complex formation. The CLIP-tag is a 20 kDa mutant of the human DNA repair protein O6-alkylguanine- DNA alkyltransferase (hAGT) that reacts specifically and rapidly with benzylcytosine (BC) derivatives, leading to covalent labeling of the CLIP-tag with a synthetic probe (Figure 1).
The CLIP-tag has a number of features that make it ideal for a variety of protein labeling applications. The rate of the reaction of the CLIP-tag with these derivatives is largely independent of the nature of the synthetic probe attached to BC, permitting the labeling of CLIP fusion proteins with a wide variety of functional groups. Many of these CLIP-tag substrates are cell-permeable, allowing live-cell imaging of protein expression and localization (Figure 2). The ability to turn on the signal at will, together with the availability of a cell-permeable nonfluorescent blocking agent (CLIP-Cell? Block), allows time-resolved pulse-chase analysis of protein trafficking. Finally, the availability of orthogonal protein labeling systems from NEB permits simultaneous labeling of multiple proteins in a single cell (SNAP-tag, another hAGT variant that reacts exclusively with O6-benzylguanine substrates, and the ACP/MCP tags, small protein tags which can be enzymatically labeled on the cell surface with Coenzyme A derivatives).
The CLIP-Cell Starter Kit contains a mammalian expression plasmid (pCLIPf) encoding the CLIP-tag flanked by restriction sites for cloning a gene of interest, and two cell-permeable fluorescent CLIP-tag substrates. A positive control plasmid (pCLIPf-H2B), encoding a CLIP-tagged protein (histone H2B) with a well-characterized nuclear localization, is also included. Lastly, a negative control “blocking agent” (CLIP-Cell Block) is included that interacts with the CLIP-tag, but is not fluorescent. There are two steps to using this system: subcloning and expression of the protein of interest as a CLIPf fusion, and labeling of the fusion with the CLIP-tag substrate of choice.