CLIP-tag™ is a novel tool for protein research, allowing the specific, covalent attachment of virtually any molecule to a protein of interest. CLIP-tag is a small protein tag based on human O6-alkylguanine-DNA-alkyltransferase (AGT). 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 link. 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 simultaneous labeling.
CLIP-tag Purified Protein can be used as a positive control for in vitro labeling with various CLIP-tag fluorescent substrates. The coding sequence of CLIP-tag was cloned into a pTXB1 derived E. coli T7 expression vector. CLIP-tag protein (MW: 20,675) was expressed and purified according to the instructions in the IMPACT™ kit manual (NEB #E6901 ). The purified CLIP-tag protein was dialyzed into 1X phosphate buffered saline (PBS) solution containing 1 mM DTT at 1 mg/ml (50 μM) and stored at -80°C.
Mass spectrometry analysis indicates the CLIP-tag purified protein has two species with molecular weights of 20,675 and 20,810 (with a C-terminal DTT moiety attached).
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