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  • CLIP-Biotin

    Description

    CLIP-Biotin is a cell-permeable substrate (BC-Biotin) based on biotin with an amidocaproyl linker. It is suitable for applications such as biotinylation of CLIP-tag™ fusion proteins in living cells for detection with streptavidin fluorophore conjugates or labeling in solution for analysis by SDS-PAGE/Western Blot or for capture with streptavidin for binding and interaction studies. This package contains 50 nmol of CLIP-Biotin substrate, sufficient to make 10 ml of a 5 μM solution for the labeling of CLIP-tag fusion proteins in cells.

    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 based on mammalian 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.

    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 fusion proteins with CLIP-Biotin is described below.
    S9221_structure_thumb
    Figure 1: Structure of CLIP-Biotin (MW 569.7 g/mol)

    Properties and Usage

    Materials Required but not Supplied

    • Cells expressing CLIP-tag proteins 
    • Tissue culture materials and media 
    • Transfection reagents 
    • DMSO

    Storage Temperature

    -20°C

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Cellular Protein Labeling (Intracellular):
      The product is tested on cells expressing the target protein intracellularly.  The intracellular target is labeled and visulaized by fluorescence microscopy
    • In Vitro Protein Labeling:

      The product is tested in an in vitro protein labeling reaction. After incubation the labeled product is visualized on SDS-PAGE by fluorescent detection and verified by mass spectrometry.

    • Physical Purity (HPLC):
      The purity of the product is determined by HPLC analysis.

    Troubleshooting

    Troubleshooting


    Troubleshooting for Labeling in vitro 

    Solubility
    If solubility problems occur with your CLIP-tag fusion protein, we recommend testing a range of pH (pH 5.0–pH 10.0) and ionic strengths. The salt concentration may also need to be optimized for your particular fusion protein (50–250 mM).

    Loss of Protein Due to Aggregation or Sticking to Tube
    If stickiness of the fusion protein is a problem we recommend adding Tween 20 at a final concentration of 0.05% to 0.1%. The CLIP-tag activity is not affected by this concentration of Tween 20.

    Incomplete Labeling
    If exhaustive labeling of a protein sample is not achieved using the recommended conditions, try the following protocol modifications: Increase the incubation time to two hours total at 25°C or to 24 hours at 4°C; or halve the volume of protein solution labeled. Both approaches may be combined. If you still have poor labeling results, we recommend checking the activity of the CLIP-tag using CLIP-Vista Green.

    If the CLIP-tag fusion has been stored in the absence of DTT or other reducing agent, or has been stored at 4°C for a prolonged period, its activity may be compromised. Include 1 mM DTT in all solutions of the CLIP-tag fusion protein, and store the fusion protein at -20°C.

    Using less than the recommended amount of substrate stock solution can significantly slow down the reaction rate.

    Loss of Activity of Protein of Interest
    If your fusion protein is particularly sensitive to degradation or to loss of activity, you can try reducing the labeling time or decreasing the labeling temperature. If you label at 4°C we recommend overnight incubation.

    Troubleshooting for Cellular Labeling 

    No Labeling

    If no labeling is seen, the most likely explanation is that the fusion protein is not expressed. Verify your transfection method to confirm that the cells contain the fusion gene of interest. If this is confirmed, check for expression of the CLIP-tag fusion protein via Western Blot using Anti-SNAP-tag Antibody (NEB #P9310). This antibody shows high crossreactivity with the CLIP-tag and can be used for Western Blot detection. Alternatively, CLIP-Vista Green (NEB #S9235) can be used to confirm presence of CLIP-tag fusion in cell extracts following SDS-PAGE, without the need for Western blotting.

    Weak Labeling
    Weak labeling may be caused by insufficient exposure of the fusion protein to the substrate. Try increasing the concentration of CLIP-tag substrate and/or the incubation time. Improving the protein expression may also improve the signal. If the protein has limited stability in the cell, it may help to analyze the samples immediately after labeling.

    Notes

    1. Storage: CLIP-Biotin should be stored at -20°C (long term) or at 4°C in the dark (short term, less than 4 weeks). Protect the substrate from light and moisture. With proper storage at -20°C the substrate should be stable for at least three years dry or 3 months dissolved in DMSO.

    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. Cellular Labeling (S9221)
    2. Labeling of Proteins in vitro (S9221)

    Selection Tools

    Troubleshooting Guides

    Application Notes

    After addition of DMSO, pipette up and down at least 10-20 times and vortex vigorously for at least one full minute to ensure full dissolution of the substrate.

    After diluting the substrate in complete medium, thoroughly pipette up and down at least 10 times to help reduce background.

    Increasing substrate concentration and/or reaction time usually results in higher background and does not necessarily increase the signal-to-background ratio (SNR).