This control plasmid contains the gene encoding the Histone H2B protein cloned upstream of the CLIPf coding sequence in pCLIPf as a fusion to the N- terminus of the CLIP-tag. Histone H2B is a member of the core histones that package DNA in the nucleus. The H2B-CLIPf fusion protein gives nuclear fluorescence when labeled with CLIP-Cell™ substrates. The full sequence and map for pCLIPf-H2B can be downloaded.
The CLIP-tag is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. The CLIP-tag is a small protein 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 the CLIPf-H2B fusion protein is described in this document. The labeling of fusion proteins with CLIP-tag substrates is described in the instructions supplied with CLIP-tag substrates.
DNASU is a central repository for plasmid clones and collections that may also be helpful.
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