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- 应用范围
- Purification (Purif)
- 原理
- Specific binding and purification of his-tagged proteins
- 品牌
- HighSpec
- 样品类型
- Cell Culture Supernatant, Cell Lysate, Tissue Lysate
- 特异性
- Affinity to His-tagged proteins
- 产品特性
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- Pre-packed column containing HighSpec INDIGO Ni-Agarose
- Novel His affinity ligand providing stability in 20 mM EDTA and 20 mM DTT
- Binding capacity up to 80 mg/mL
- Suitable for high flow rates on FPLC instruments
- pH tolerant from pH 4-9.
- Delivered as 5 x prepacked cartridges containing 1 mL bed volume each.
- 组件
- Pre-packed affinity cartridges
- 试剂未包括
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- FPLC instrument Lysis
- Wash
- Elution Buffers
- Ice bath
- Refrigerated centrifuge for 50 mL tube (min 10,000 x g)
- 50 mL centrifuge tube
- Micropipettor and Micropipetting tips
- pH meter
- End-over-end shaker
- SDS-PAGE buffers, reagents and equipment
Optional: Western Blot reagents and equipment
- Bead Ligand
- Ni-INDIGO
- Bead Matrix
- Agarose beads
- Bead Size
- 100 μm
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Magnetic Concanavalin A Beads (Agarose)
CUT&RUN, CUT&Tag, Sep Concanavalin A Magnetic Agarose beads 30 μm
Ni-NTA MagBeadsPull-Down, Purif, Sep Ni-NTA 25 µm
Ni-NTA AgarosePurif, Sep Ni-NTA 40 µm
Rho1D4 AgarosePurif, Sep Rho1D4 40 µm
BFP-CatcherCo-IP, IP, Purif, ChIP, RIP Antibody Agarose beads 90 μm
INDIGO Ni-AgarosePurif Ni-INDIGO Agarose beads 100 μm
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- 应用备注
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Technical features of Cartridges:
- Bed volume: 1 mL
- Dimensions (mm): 6.2 X 50
- Body material: Polypropylene
- Inlet: 10-32 UNF female thread
- Outlet:10-32 UNF female thread - 说明
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Sample Volume for an assay: >200 mL E.coli culture volume or corresponding quantity. Protocol can be scaled up easily.
- 样本量
- 200 mL
- 实验时间
- 4 - 5 h
- 实验流程
- Purification of his-tagged protein on FPLC instruments
- 试剂准备
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A Purification under native conditions:
- Native Lysis buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 10 mM, pH 8
Optional: Add 1 tablet protease inhibitor cocktail to the Lysis Buffer.
Tip: Lysis Buffer contains 10 mM imidazole to prevent binding of untagged proteins. If His-tagged proteins do not bind under these conditions, reduce the imidazole concentration to 1-5 mM. - Native Wash buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 20 mM, pH 8
- Native Elution buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 250- 500 mM, pH 8
Additional chemicals required: Lysozyme, Benzonase® nuclease,
Optional: Protease inhibitor cocktail
B Purification under denaturing conditions:- Denaturing Lysis buffer: NaH2PO4 100 mM, Tris base 10 mM, Urea 8 M, pH 8.0,
Optional: Benzonase® nuclease (e.g. Merck Milipore, #707464) - Denaturing Wash buffer: NaH2PO4 100 mM, Tris base 10 mM, Urea 8 M, pH 6.3NaH2PO4 100 mM
- Denaturing Elution buffer: NaH2PO4 100 mM, Tris base 10 mM, Urea 8 M, pH 4.5
Tip: If the target protein is acid-labile, elution can be performed with 250-500 mM imidazole.
Note: Due to urea dissociation, adjust the pH immediately before use.
- Native Lysis buffer: NaH2PO4 50 mM, NaCl 300 mM, Imidazole 10 mM, pH 8
- 实验流程
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Standard protocols for His affinity purification can be applied. Please also refer to the FPLC instrument manufacturer's instructions.
- 结果分析
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Analyze by SDS-PAGE, Bradford Assay or spectrophotometrically.
- 限制
- 仅限研究用
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- 储存条件
- 4 °C
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