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  • pCLIPf-Cox8A Control Plasmid

    Discontinued Date

    01/01/2013
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    Discontinued Products

    Description

    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.

    Figure 1: Live HEK293 cells transiently transfected with pCLIPf-Cox8A. Cells were labeled with CLIP-Cell TMR-Star (red) for 30 minutes and counterstained with Hoechst 33342 (blue).


    N9217b_thumb

    Properties and Usage

    Materials Required but not Supplied

    Cell culture media and reagents
    Mammalian cell lines
    Transfection reagents
    CLIP-tag substrates

    Storage Temperature

    -20°C

    Notes

    1. Storage: pCLIPf-Cox8A is supplied in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) at a concentration of 0.5 µg/µl. Plasmid solutions can be stored at 4°C for up to one week. For long-term storage -20°C is recommended.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.
    1. Cellular Imaging and Analysis FAQs
    1. Expression of CLIPf-Cox8A (N9217)

    Selection Tools

    Troubleshooting Guides

    Interactive Tools

    Application Notes

    If you generate more plasmid DNA by bacterial transformation, we recommend isolating the plasmid DNA using an endotoxin-free plasmid prep kit prior to transfection into mammalian cells.