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  • pCLIPf-H2B Control Plasmid

    Description

    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.

    Figure 1: Live CHO-K1 cells transiently transfected with pCLIPf-H2B. Cells were labeled with CLIP-Cell™ 505 (green) for 30 minutes and counterstained with Hoechst 33342 (blue).

    DNASU is a central repository for plasmid clones and collections that may also be helpful.



    N9218b

    Properties and Usage

    Materials Required but not Supplied

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

    Storage Temperature

    -20°C

    Notes

    1. Storage: pCLIPf-H2B 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.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Cellular Imaging and Analysis FAQs
    1. Expression of CLIPf-H2B (N9218)

    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.