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GFP-Catcher (agarose magnetic beads)

RIP, Co-IP, IP, Purif, ChIP Antibody Magnetic Agarose beads 90 μm
Catalog No. ABIN7272855
  • Key Features
    • High-affinity anti-GFP single-domain antibody (sdAb) covalently bound to 4 % cross-linked agarose magnetic beads with 50-150 µm diameter.
    • The Alpaca sdAb clone 1H1 is specific for GFP and many GFP derivatives. Capacity >4 µg GFP / 1 µL packed beads (2 µL bead slurry).
    • Compatible with physiological buffers, common Lysis and Wash Buffers, and high stringency buffers.
    Target See all GFP products
    GFP (Green Fluorescent Protein (GFP))
    Reactivity
    Aequorea victoria
    Expression System
    E.coli
    Application
    RNA-Binding Protein Immunoprecipitation (RIP), Protein Complex Immunoprecipitation (Co-IP), Immunoprecipitation (IP), Purification (Purif), Chromatin Immunoprecipitation (ChIP)
    Purpose
    GFP-Catcher is based on a high-affinity single-domain antibody (sdAb) that is covalently immobilized on 4% cross-linked magnetic agarose.
    Specificity
    GFP (green fuorescent protein) and common GFP derivatives like EGFP, mEGFP, Sirius, tSapphire, Cerulean, eCFP, mTurquoise, acGFP, Emerald, superecliptic pHluorin, paGFP, superfolder GFP, eYFP, mVenus and Citrine. Other not tested.
    No Cross-Reactivity
    Does not cross-react with mCherry, dsRed, mRFP, mTagBFP or their most common derivatives.
    Characteristics
    GFP-Catcher is based on a high-affinity alpaca single-domain antibody (sdAb) that is covalently immobilized on 4 % cross-linked magnetic agarose beads. The innovative, oriented and selective attachment via a flexible linker guarantees a high accessibility of the sdAbs and largely eliminates batch-to-batch variations. Due to the single-chain nature of sdAbs and their covalent attachment, no "leakage" of light and heavy chains from IgGs is observed during elution with SDS sample buffer.
    GFP-Catcher thus features high affinity and superior capacity for GFP fusion proteins while showing negligible non-specific background.
    GFP-Catcher immobilizes a wide range of GFP derivatives.
    GFP-Catcher is compatible not only with physiological buffers but also with high stringency buffers.
    GFP-Catcher thus provides great freedom to adjust the binding and washing conditions to the experimental needs.
    Material not included
    wash buffers, columns, tubes
    Bead Ligand
    Antibody
    Bead Matrix
    Magnetic Agarose beads
    Bead Size
    90 μm
    Product Specific Information

    Our GFP Catcher is a product based on the GFP pull-down technique used to isolate GFP fusion proteins from cell lysates. It consists of special magnetic beads (product ABIN7272855) or agarose beads (product ABIN5311508) coated with an antibody against GFP. These beads allow efficient and selective binding to GFP or GFP-fused target proteins.

    The application of GFP Catcher is similar to the GFP pull-down method. First, the protein of interest is expressed in the cells with it fused to GFP. Then, the cell lysate is prepared to extract the target proteins. The GFP catcher beads are then added to the cell lysate and bound to these proteins by specifically binding to the GFP or GFP fusion protein.

    After the beads with the bound target proteins are isolated, thorough purification is performed to remove components that are not specifically bound. Finally, the target proteins can be separated from the beads and used for further analysis such as immunoblotting or mass spectrometry.

    GFP Catcher offers the advantage of quick and easy isolation of GFP fusion proteins. It is a useful tool for protein analysis and allows efficient identification of proteins interacting with GFP fusion protein. GFP Catcher is widely used in cell biology research to study protein-protein interactions and deepen the understanding of cellular processes.

  • Comment

    4% cross-linked magnetic agarose (bead size 50-150 μm) with covalently immobilized single-domain antibody

    Protocol

    This protocol provides a general outline of how to use GFP-Catcher (agarose magnetic beads) for immunoprecipitation using a microcentrifuge for sedimentation. Alternatively, it is possible to use GFP-Catcher agarose beads in spin columns. All protocol steps should be carried out at 4 °C.

    Protocol as PDF

    1. For mammalian cells, harvest 106-108 cells per sample.
    2. Lyse cells according to established protocols in 0.2 to 1.5 mL volume. Recommended Buffer Conditions: GFP-Catcher resins are compatible with commonly used Lysis and Washing buffers, e.g. RIPA buffer. The following buffer conditions have been tested:
      • pH ranging from pH 5 to pH 9
      • 2 % Triton X-100, 1 % Tween-20, 1 % NP-40, 1 % CHAPS, 1 % Deoxycholate, 0.1 % SDS
      • 4 M NaCl, 2 M KCl, 1 M MgCl2
      • 100 mM EDTA
      • 4 M urea
      • 10 mM DTT, 10 mM 2-Mercaptoethanol
      • Protease Inhibitors
      • RNAse A, DNAse I, Benzonase
    3. Centrifuge cell lysates in microcentrifuge tubes for 10 min at 14.000 x g at 4 °C. Keep a small samples as “input” fraction.
    4. Transfer the supernatant to a fresh microcentrifuge tube for each sample and keep at 4 °C.
    5. Homogenize the GFP-Catcher (agarose magnetic beads) slurry gently by shaking.
    6. Transfer 20 μL bead slurry to a 1.5 mL microcentrifuge tube for each sample.
    7. Add 1 mL Lysis Buffer to equilibrate GFP-Catcher (agarose magnetic beads).
    8. Place the tubes on a magnet stand until the fluid is clear. Remove the supernatant carefully.
    9. Repeat wash steps once for a total of two washes.
    10. Resuspend equilibrated GFP-Catcher (agarose magnetic beads) gently with the cell lysate supernatant.
    11. Rotate the microcentrifuge tubes for 1 h at 4 °C.
    12. Place the tubes on a magnet stand until the fluid is clear. Keep a small aliquot of the supernatant as “unbound” fraction. Carefully remove the supernatant.
    13. Resuspend GFP-Catcher (agarose magnetic beads) in 1 mL Lysis Buffer.
    14. Place the tubes on a magnet stand until the fluid is clear and carefully remove the supernatant.
    15. Repeat wash steps twice for a total of three washes.
    16. Resuspend GFP-Catcher (agarose magnetic beads) gently in 1 mL TBS.
    17. Place the tubes on a magnet stand until the fluid is clear and carefully remove the supernatant.
    18. Repeat wash steps once for a total of two washes.
    19. Resuspend GFP-Catcher (agarose magnetic beads) resin in 50 µL 2X SDS samples buffer.
    20. Heat GFP-Catcher (agarose magnetic beads) resin for 5 min to 95 °C.
    21. Place the tubes on a magnet stand until the fluid is clear and transfer the supernatant to fresh microcentrifuge tubes. Keep the GFP-Catcher (agarose magnetic beads) as backup.
    Restrictions
    For Research Use only
  • Buffer
    50 % slurry in PBS containing 20 % Ethanol
    Storage
    4 °C
    Storage Comment
    Store at 4°C for up to 12 months. Do not freeze!
    Expiry Date
    12 months
  • Target
    GFP (Green Fluorescent Protein (GFP))
    Alternative Name
    GFP (GFP Products)
    Synonyms
    green fluorescent protein Bead, gfp Bead
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