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GFP-Catcher

RIP, Co-IP, IP, Purif, ChIP Antibody Agarose beads 90 μm
Produktnummer ABIN5311508
  • Highlights
    • High-affinity anti-GFP single-domain antibody (sdAb) covalently bound to 4 % cross-linked agarose 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 Alle GFP Produkte
    GFP (Green Fluorescent Protein (GFP))
    Reaktivität
    Aequorea victoria
    Expressionssystem
    E.coli
    Applikation
    RNA-Binding Protein Immunoprecipitation (RIP), Protein Complex Immunoprecipitation (Co-IP), Immunoprecipitation (IP), Purification (Purif), Chromatin Immunoprecipitation (ChIP)
    Verwendungszweck
    GFP-Catcher is based on a high-affinity single-domain antibody (sdAb) that is covalently immobilized on 4% cross-linked agarose beads.
    Proben
    Cell Extracts
    Spezifität
    Recognizes GFP (green fluorescent protein) and common GFP derivatives like EGFP, mEGFP, Sirius, tSapphire, Cerulean, eCFP, mTurquoise, acGFP, Emerald, superecliptic pH luorin, paGFP, superfolder GFP, eYFP, mVenus and Citrine and most common CFP and YFP variants.
    Kreuzreaktivität (Details)
    Does not cross-react with mCherry, mRFP, dsRed, mTagBFP or their most common derivatives.
    Produktmerkmale
    GFP-Catcher is based on a high-affinity single-domain antibody (sdAb) that is covalently immobilized on 4 % cross-linked 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.
    Bestandteile
    4 % cross-linked agarose (bead size 50-150 μm) with covalently immobilized single-domain antibody
    Benötigtes Material
    wash buffers, columns, tubes
    Bead Ligand
    Antibody
    Bead Matrix
    Agarose beads
    Bead Size
    90 μm
    Produktspezifische Information

    Unser GFP Catcher ist ein Produkt, das auf der GFP Pull-Down-Technik basiert und zur Isolierung von GFP-Fusionsproteinen aus Zelllysaten verwendet wird. Es besteht aus speziellen magnetischen Beads (Produkt ABIN7272855) bzw. Agarose Beads (Produkt ABIN5311508), die mit einem Antikörper gegen GFP beschichtet sind. Diese Beads ermöglichen eine effiziente und selektive Bindung an GFP oder GFP-fusionierte Zielproteine.

    Die Anwendung von GFP Catcher ist ähnlich wie bei der GFP Pull-Down-Methode. Zunächst wird das zu untersuchende Protein in den Zellen exprimiert, wobei es mit GFP fusioniert ist. Anschließend wird das Zelllysat hergestellt, um die Zielproteine zu extrahieren. Die GFP Catcher-Beads werden dann in das Zelllysat gegeben und durch die spezifische Bindung an das GFP oder das GFP-Fusionsprotein an diese Proteine gebunden.

    Nachdem die Beads mit den gebundenen Zielproteinen isoliert wurden, erfolgt eine gründliche Reinigung, um nicht spezifisch gebundene Komponenten zu entfernen. Schließlich können die Zielproteine von den Beads abgetrennt und für weitere Analysen wie Immunoblotting oder Massenspektrometrie verwendet werden.

    GFP Catcher bietet den Vorteil einer schnellen und einfachen Isolierung von GFP-Fusionsproteinen. Es ist ein nützliches Werkzeug für die Proteinanalyse und ermöglicht die effiziente Identifizierung von Proteinen, die mit dem GFP-Fusionsprotein in Wechselwirkung treten. GFP Catcher wird häufig in der zellbiologischen Forschung eingesetzt, um Protein-Protein-Interaktionen zu untersuchen und das Verständnis der zellulären Prozesse zu vertiefen.

  • Protokoll

    This protocol provides a general outline of how to use GFP-Catcher (agarose 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 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 beads).
    8. Centrifuge GFP-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
    9. Repeat wash steps once for a total of two washes.
    10. Resuspend equilibrated GFP-Catcher (agarose beads) gently with the cell lysate supernatant.
    11. Rotate the microcentrifuge tubes for 1 h at 4 °C.
    12. Centrifuge microcentrifuge tubes for 1 min at 1000 x g at 4 °C. Keep a small sample as “unbound” fraction. Carefully remove the supernatant.
    13. Resuspend GFP-Catcher (agarose beads) in 1 mL Lysis Buffer.
    14. Centrifuge GFP-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
    15. Repeat wash steps twice for a total of three washes.
    16. Resuspend GFP-Catcher (agarose beads) gently in 1 mL TBS.
    17. Centrifuge GFP-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
    18. Resuspend GFP-Catcher (agarose beads) gently in 1 mL TBS.
    19. Centrifuge GFP-Catcher (agarose beads) for 1 min at 3000 x g and carefully remove the supernatant.
    20. Resuspend GFP-Catcher (agarose beads) resin in 50 µL 2X SDS samples buffer.
    21. Heat GFP-Catcher (agarose beads) resin for 5 min to 95 °C.
    22. Centrifuge microcentrifuge tubes for 1 min at 3000 x g and transfer the supernatant to fresh microcentrifuge tubes. Keep the GFP-Catcher (agarose beads) as backup.

    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Buffer
    50 % slurry in PBS containing 20 % Ethanol
    Lagerung
    4 °C
    Informationen zur Lagerung
    Store at 4 °C, do not freeze
    Haltbarkeit
    12 months
  • Target
    GFP (Green Fluorescent Protein (GFP))
    Andere Bezeichnung
    GFP (GFP Produkte)
    Synonyme
    green fluorescent protein Bead, gfp Bead
    Substanzklasse
    Viral Protein
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