CLIP-Cell™ 360 is a blue fluorescent substrate that can be used to label CLIP-tag™ fusion proteins inside living cells and in vitro. This cell-permeable substrate (BC-360) is based on aminomethylcoumarin and is suitable for DAPI filter sets or appropriate blue lasers. It has an excitation maximum at 357 nm and emission maximum at 437 nm. This package includes 50 nmol of CLIP-Cell 360 substrate, sufficient to make 10 ml of a 5 µM CLIP-tag fusion protein labeling solution.
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-DNAalkyltransferase (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.
There are two steps to using this system: subcloning and expression of the protein of interest as a CLIP-tag fusion, and labeling of the fusion with the CLIP-tag substrate of choice. Expression of CLIP-tag fusion proteins is described in the documentation supplied with CLIP-tag plasmids. The labeling of the fusion proteins with the CLIP-tag substrate is described in this document.
Materials Required but not Supplied
• Cells expressing CLIP-tag fusion proteins
• Tissue culture materials and media
• Transfection reagents
• Fluorescence microscope with suitable filter set
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