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  • SNAP-Capture Pull-Down Resin

    Description

    The SNAP-Capture Pull Down Resin is used to selectively capture and immobilize a SNAP-tag® fusion protein from solution using agarose beads. These beads have a high loading capacity for SNAP-tag fusion proteins and show very low non-specific absorption of proteins from a complex lysate, making them especially suitable for pull down applications. The SNAP-Capture Pull Down Resin is prepared by the coupling of SNAP-tag substrate benzylguanine with a highly cross-linked agarose (4%) with excellent physical characteristics. Two ml of SNAP-Capture Pull Down Resin is sufficient to perform 25 pull down assays using 80 µl of resin suspension per assay. 

    The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. The SNAPtag is based on human O6-alkylguanine-DNAalkyltransferase (hAGT). SNAP-tag substrates are fluorophores, biotin or beads conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.

    There are two steps to the use of this system: sub cloning and expression of the protein of interest as a SNAP-tag fusion, and capture and immobilization of the fusion protein using SNAP-Capture Pull Down Resin.

    Figure 1. Substrate structure on SNAP-Capture Pull Down Resin

    Properties and Usage

    Materials Required but not Supplied

    • Protein sample containing the protein to immobilize expressed as a SNAP-tag fusion 
    • Buffer for immobilization and washing 
    • Microcentrifuge

    Storage Temperature

    4°C

    Binding Capacity

    SNAP-Capture Pull Down Resin (80 µl) was washed, incubated with 200 µl of 1 mg/ml SNAP-tag CBD (Chitin Binding Domain) for 1 hour at room temperature, then rewashed as described in these instructions. Binding capacity was determined to be 3 mg/ml bed resin.

    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.
    • Binding Capacity (Affinity Tagged Bead/Resin):
      The product is tested by incubation with the appropriate affinity tagged protein and the binding capacity determined

    Notes

    1. Storage of Pull Down Resin: Do not freeze the SNAP-Capture Pull Down Resin. Store the unused resin at 4°C. With proper storage, resin should be stable for at least Two years.
    2. Correct storage and handling of SNAP-tag fusion proteins is essential to maintain reactivity of the SNAP-tag prior to immobilization.
    3. SNAP-tag fusion proteins can be purified before immobilization, but the immobilization reaction also works in non-purified protein solutions including cell lysates.
    4. Add 1 mM DTT to buffers used for the storage of SNAP-tag fusion proteins. Protein samples should be stored at -20°C, or at -80°C for long-term storage. Handling at temperatures above 0°C should be minimized by thawing the SNAP-tag protein samples shortly before use, and keeping them on ice until just before the immobilization.
    5. If a particular fusion protein requires buffers without reducing agents, pay particular attention to minimize all handling steps of the protein above 4°C before the labeling reaction.
    6. The SNAP-tag itself is tolerant of a wide range of buffers. The requirements of your fusion partner should dictate the selection of the buffer. The following storage buffer composition is recommended, especially when freezing protein material: pH between 7.0 and 8.0, monovalent salts (e.g. sodium chloride) between 50 mM and 250 mM, and at least 1 mM DTT. Non-ionic detergents can be added if required, but ionic detergents should be avoided because they reduce the activity of the SNAP-tag. Many proteins benefit from the addition of glycerol for frozen storage, typically 20% v/v.
    7. Immobilized SNAP-tag fusion proteins should be stored at 4°C. Sodium azide may be added to 2 mM final concentration to prevent bacterial growth. Depending on the stability of the fusion partner, under these conditions the immobilized protein should be stable at 2–6°C for several months. The SNAP-Capture Pull Down Resin should not be frozen.
    8. The SNAP-tag fusion protein is linked to the SNAP-Capture Pull Down Resin by a covalent bond. Therefore the immobilized protein is essentially irreversibly bound to the resin. It is important however to preserve the functional stability of the protein fused to the SNAP-tag as much as possible. We recommend handling the immobilized fusion protein and storing between use at 4°C, to prepare it just before use, and to handle it as gently as possible.

    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. Use SNAP-Capture Pull Down Resin (S9144)

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