This control plasmid contains the gene encoding the Cytochrome C oxidase, subunit 8-2 (COX8-2) protein cloned upstream of the CLIPf coding sequence in pCLIPf, as a fusion to the N- terminus of the CLIP-tag. Cytochrome C oxidase is located in the inner mitochondrial membrane and is the terminal enzyme of the respiratory chain. The COX8-2-CLIPf fusion protein gives mitochondrial fluorescence when labeled with CLIP-Cell™ substrates. The full sequence and map for pCLIPf-Cox8A can be downloaded.
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-Cox8A 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. Expressing the CLIPf-Cox8A 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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