mNeonGreen-Catcher
RIP, ChIP, IP, Co-IP, Purif
Antibody
Agarose beads
90 μm
-
- 抗原
- mNeonGreen
- 适用
- Branchiostoma lanceolatum
- 应用范围
- RNA-Binding Protein Immunoprecipitation (RIP), Chromatin Immunoprecipitation (ChIP), Immunoprecipitation (IP), Protein Complex Immunoprecipitation (Co-IP), Purification (Purif)
- 原理
- mNeonGreen-Catcher is based on a high-affinity single-domain antibody (sdAb) that is covalently immobilized on 4% cross-linked agarose beads.
- 特异性
- Recognizes mNeonGreen.
- 交叉反应 （详细）
- Does not cross-react with any GFP-, dsRed, or TagBFP derivatives.
- 产品特性
-
mNeonGreen-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.
mNeonGreen-Catcher thus features high affinity and superior capacity for mNeonGreen fusion proteins while showing negligible non-specific background.
mNeonGreen-Catcher is compatible not only with physiological buffers but also with high stringency buffers.
mNeonGreen-Catcher thus provides great freedom to adjust the binding and washing conditions to the experimental needs. - Bead Ligand
- Antibody
- Bead Matrix
- Agarose beads
- Bead Size
- 90 μm
-
- 应用备注
- Capacity: > 3 μg mNeonGreen per μl of packed beads
- 实验流程
-
This protocol provides a general outline of how to use mNeonGreen-Catcher (agarose beads) for immunoprecipitation using a microcentrifuge for sedimentation. Alternatively, it is possible to use mNeonGreen-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: mNeonGreen-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 mNeonGreen-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 mNeonGreen-Catcher (agarose beads).
- Centrifuge mNeonGreen-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 mNeonGreen-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 mNeonGreen-Catcher (agarose beads) in 1 mL Lysis Buffer.
- Centrifuge mNeonGreen-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 mNeonGreen-Catcher (agarose beads) gently in 1 mL TBS.
- Centrifuge mNeonGreen-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
- Resuspend mNeonGreen-Catcher (agarose beads) gently in 1 mL TBS.
- Centrifuge mNeonGreen-Catcher (agarose beads) for 1 min at 3000 x g and carefully remove the supernatant.
- Resuspend mNeonGreen-Catcher (agarose beads) resin in 50 µL 2X SDS samples buffer.
- Heat mNeonGreen-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 mNeonGreen-Catcher (agarose beads) as backup.
- 限制
- 仅限研究用
-
- 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
-
- 抗原
- mNeonGreen
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