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RFP-Catcher

Co-IP, IP, Purif, ChIP, RIP Antibody Agarose beads 90 μm
N° du produit ABIN5311510
  • Highlights
    • High-affinity single-domain antibody
    • Recognizes mRFP, mCherry, mScarlet-i, tdTomato, dsRed etc.
    • Bead size 50-150 μm
    Antigène Tous les produits RFP
    RFP (Red Fluorescent Protein (RFP))
    Reactivité
    Discosoma
    Expression System
    E.coli
    Application
    Protein Complex Immunoprecipitation (Co-IP), Immunoprecipitation (IP), Purification (Purif), Chromatin Immunoprecipitation (ChIP), RNA-Binding Protein Immunoprecipitation (RIP)
    Fonction
    RFP-Catcher is based on a high-affinity single-domain antibody (sdAb) that is covalently immobilized on 4% cross-linked agarose beads.
    Type d'échantillon
    Cell Extracts
    Specificité
    Recognizes most common red fluorescent proteins like mRFP and derivatives like mCherry, mScarlet-i, tdTomato, dsRed and mOrange.
    Réactivité croisée (Details)
    Does not cross-react with GFP or mTagBFP derivatives.
    Attributs du produit
    RFP-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.
    RFP-Catcher thus features high affinity and superior capacity for RFP fusion proteins while showing negligible non-specific background.
    RFP-Catcher immobilizes a wide range of RFP derivatives including mCherry and mScarlet.
    RFP Selector is compatible not only with physiological buffers but also with high stringency buffers.
    RFP-Catcher thus provides great freedom to adjust the binding and washing conditions to the experimental needs.
    Ingrédients
    4 % cross-linked agarose (bead size 50-150 μm) with covalently immobilized single-domain antibody
    Matériel non inclus
    wash buffers, columns, tubes
    Bead Ligand
    Antibody
    Bead Matrix
    Agarose beads
    Bead Size
    90 μm
  • Indications d'application
    Coating: sdAb anti-RFP clone 2B12
    Matrix: 4 % cross-linked agarose, bead size 50-150 μm
    Capacity: > 4 μg RFP per μl of packed beads (= 2 μL of slurry)
    Buffer Compatibility:
    • Common buffer substances at pH 5 to 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
    • RNAse A, DNAse I, Benzonase, protease inhibitors
    Protocole

    This protocol provides a general outline of how to use RFP-Catcher (agarose beads) for immunoprecipitation using a microcentrifuge for sedimentation. Alternatively, it is possible to use RFP-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: RFP-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 RFP-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 RFP-Catcher (agarose beads).
    8. Centrifuge RFP-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 RFP-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 RFP-Catcher (agarose beads) in 1 mL Lysis Buffer.
    14. Centrifuge RFP-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 RFP-Catcher (agarose beads) gently in 1 mL TBS.
    17. Centrifuge RFP-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
    18. Resuspend RFP-Catcher (agarose beads) gently in 1 mL TBS.
    19. Centrifuge RFP-Catcher (agarose beads) for 1 min at 3000 x g and carefully remove the supernatant.
    20. Resuspend RFP-Catcher (agarose beads) resin in 50 µL 2X SDS samples buffer.
    21. Heat RFP-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 RFP-Catcher (agarose beads) as backup.

    Restrictions
    For Research Use only
  • Buffer
    50 % slurry in PBS containing 20 % Ethanol
    Stock
    4 °C
    Stockage commentaire
    Store at 4 °C, do not freeze
    Date de péremption
    12 months
  • Antigène
    RFP (Red Fluorescent Protein (RFP))
    Autre désignation
    RFP (RFP Produits)
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