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- Key Features
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- High-affinity single-domain antibody
- Recognizes mRFP, mCherry, mScarlet-i, tdTomato, dsRed etc.
- Bead size 50-150 μm
- 抗原 See all RFP products
- RFP (Red Fluorescent Protein (RFP))
- 适用
- Discosoma
- 应用范围
- Protein Complex Immunoprecipitation (Co-IP), Immunoprecipitation (IP), Purification (Purif), Chromatin Immunoprecipitation (ChIP), RNA-Binding Protein Immunoprecipitation (RIP)
- 原理
- RFP-Catcher is based on a high-affinity single-domain antibody (sdAb) that is covalently immobilized on 4% cross-linked agarose beads.
- 样品类型
- Cell Extracts
- 特异性
- Recognizes most common red fluorescent proteins like mRFP and derivatives like mCherry, mScarlet-i, tdTomato, dsRed and mOrange.
- 交叉反应 （详细）
- Does not cross-react with GFP or mTagBFP derivatives.
- 产品特性
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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. - 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
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- 应用备注
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Coating: sdAb anti-RFP clone 2B12
Matrix: 4 % cross-linked agarose, bead size 50-150 μm
Capacity: > 3 μg RFP per μl of packed beads
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
- 实验流程
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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.
- For mammalian cells, harvest 106-108 cells per sample.
- 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
- Centrifuge cell lysates in microcentrifuge tubes for 10 min at 14.000 x g at 4 °C. Keep a small samples as “input” fraction.
- Transfer the supernatant to a fresh microcentrifuge tube for each sample and keep at 4 °C.
- Homogenize the RFP-Catcher (agarose beads) slurry gently by shaking.
- Transfer 20 μL bead slurry to a 1.5 mL microcentrifuge tube for each sample.
- Add 1 mL Lysis Buffer to equilibrate RFP-Catcher (agarose beads).
- Centrifuge RFP-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
- Repeat wash steps once for a total of two washes.
- Resuspend equilibrated RFP-Catcher (agarose beads) gently with the cell lysate supernatant.
- Rotate the microcentrifuge tubes for 1 h at 4 °C.
- Centrifuge microcentrifuge tubes for 1 min at 1000 x g at 4 °C. Keep a small sample as “unbound” fraction. Carefully remove the supernatant.
- Resuspend RFP-Catcher (agarose beads) in 1 mL Lysis Buffer.
- Centrifuge RFP-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
- Repeat wash steps twice for a total of three washes.
- Resuspend RFP-Catcher (agarose beads) gently in 1 mL TBS.
- Centrifuge RFP-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
- Resuspend RFP-Catcher (agarose beads) gently in 1 mL TBS.
- Centrifuge RFP-Catcher (agarose beads) for 1 min at 3000 x g and carefully remove the supernatant.
- Resuspend RFP-Catcher (agarose beads) resin in 50 µL 2X SDS samples buffer.
- Heat RFP-Catcher (agarose beads) resin for 5 min to 95 °C.
- 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.
- 限制
- 仅限研究用
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- 缓冲液
- 50 % slurry in PBS containing 20 % Ethanol
- 储存条件
- 4 °C
- 储存方法
- Store at 4 °C, do not freeze
- 有效期
- 12 months
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- 抗原
- RFP (Red Fluorescent Protein (RFP))
- 别名
- RFP (RFP 产品)
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