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GST-Catcher

Co-IP, IP, Purif, ChIP, RIP Antibody Agarose beads 90 μm
产品编号 ABIN5311506
发货至: 中国
  • 抗原 See all GST products
    GST (Glutathione S Transferase (GST))
    适用
    Schistosoma japonicum
    Expression System
    E.coli
    应用范围
    Protein Complex Immunoprecipitation (Co-IP), Immunoprecipitation (IP), Purification (Purif), Chromatin Immunoprecipitation (ChIP), RNA-Binding Protein Immunoprecipitation (RIP)
    样品类型
    Cell Extracts
    特异性
    The Antibody with Clone G19 recognizes Schistosoma japonicum Glutathione S-transferase (GST)
    产品特性
    GST-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. GST-Catcher thus features high affinity and superior capacity for GST fusion proteins while showing negligible non-specific background. GST-Catcher is compatible not only with physiological buffers but also with high stringency buffers. GST-Catcher thus provides great freedom to adjust the binding and washing conditions to the experimental needs.
    组件
    4 % cross-linked agarose (bead size 50-150 μm) with covalently immobilized single-domain antibody
    试剂未包括
    wash buffers, columns, tubes
    Bead Ligand
    Antibody
    Bead Matrix
    Agarose beads
    Bead Size
    90 μm
  • 应用备注
    200 μL slurry for up to 10 reactions
    说明

    Capacity: > 4 μg GST per μl of packed beads (i.e. 2 μl of slurry)

    实验流程

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

    限制
    仅限研究用
  • 缓冲液
    50 % slurry in 20 % ethanol / PBS 0.09 % sodium azide
    储存液
    Sodium azide
    注意事项
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    储存条件
    4 °C
  • 抗原
    GST (Glutathione S Transferase (GST))
    别名
    Glutathione S-transferase (GST) (GST 产品)
    别名
    GST Bead, GST 13-13 Bead, GST13 Bead, GST13-13 Bead, GSTK1-1 Bead, hGSTK1 Bead, 1500002K10Rik Bead, Gst Bead, Ptgds2 Bead, glutathione S-transferase kappa 1 Bead, microsomal glutathione S-transferase 1 Bead, hematopoietic prostaglandin D synthase Bead, GSTK1 Bead, Mgst1 Bead, Hpgds Bead
    背景
    Glutathione S-transferase (GST) from Schistosoma japonicum is a 26 kDa enzyme, often found as a fusion partner in expression systems. GST folds rapidly and boosts the expression and the folding of the fusion partner. It is also used as purification tag wi
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