This control plasmid contains the gene encoding the seven-pass transmembrane protein Neurokinin-1 receptor (NK1R), a member of the G-protein coupled receptor family. The NK1R was cloned downstream of the CLIPf coding sequence in pCLIPf, as a fusion to the C- terminus of the CLIPf.
An import signal sequence for import into the endoplasmic reticulum (ER) was cloned upstream of the CLIPf. This import signal sequence is based on the serotonin receptor 5HT3A. The CLIPf-neurokinin-1 receptor is inserted in the plasma membrane with the CLIPf exposed to the extracellular side of the membrane. The CLIPf-NK1R fusion protein gives plasma membrane labeling when labeled with cell permeable CLIP-Cell™ substrates and non-cell permeable CLIP-Surface substrates.
The CLIP-tag™ protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. CLIP-tag is a protein tag based on human O6-alkylguanine-DNA-alkyltransferase (hAGT). CLIP-tag substrates are derivatives of benzylcytosine (BC). In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the reactive cysteine of CLIP-tag forming a stable thioether bond. Although CLIP-tag is based on the same protein as SNAP-tag, the benzylcytosine substrates form a separate class of substrates, different from the benzylguanine substrates recognized by SNAP-tag. CLIP-tag and SNAP-tag® can be used for orthogonal and complementary labeling of two proteins simultaneously in the same cells.
pCLIPf contains an improved version of CLIP-tag, termed CLIPf. CLIPf displays faster kinetics in in vitro labeling and fast, specific and efficient labeling in live and fixed cell applications, thereby rendering it a desired research tool for analysis of protein dynamics. There are two steps to using this system: sub cloning and expression of the protein of interest as a CLIPf fusion, and labeling of the fusion with the CLIP-tag substrate of choice. Expression of CLIPf-NK1R fusion protein is described in this document. The labeling of the fusion proteins with CLIP-tag substrates is described in the instructions supplied with CLIP-tag substrates.
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