Generation of Polyclonal Specific Antibodies
制备特异性多克隆抗体
发布: 2013年06月05日第3卷第11期 DOI: 10.21769/BioProtoc.779 浏览次数: 11811
评审: Ru Zhang
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参见作者原研究论文
The authors used this protocol in:
Mar 2013
Abstract
Generation of antibodies specific for a protein of interest is a common method in many disciplines. This protocol details the steps in production of a polyclonal antibody in rabbits using a bacterially expressed fusion protein as an antigen. The protocol is generated based on data presented in Wirschell et al.(2013).
Keywords: Antibody (抗体)
Dynein (动力蛋白)
Cilia (纤毛)
Flagella (鞭毛)
Protein purification (蛋白纯化)
Materials and Reagents
- Topo TA cloning kit with One Shot competent cells (Life Technologies, Invitrogen™, catalog number: K457501 )
- pCR2.1 TOPO
- pET28a
- pMal-C (New England Biolabs)
- BL21 (DE3) pLysS competent cells (Stratagene, catalog number: 200132 )
- Bacto agar (BD Biosciences, catalog number: 214530 )
- Bacto Peptone Peptone (BD Biosciences, catalog number: 211677 )
- Bacto yeast extract (BD Biosciences, catalog number: 212750 )
- NaCl (Thermo Fisher Scientific, catalog number: 7647-14-5 )
- IPTG (Promega Corporation, catalog number: PR-V3953 )
- Novagen BugBuster Lysis buffer (Thermo Fisher Scientific, catalog number: 50-230-9216 )
- Urea (Thermo Fisher Scientific, catalog number: U15-500 )
- Novagen His-Bind Purification kit (Thermo Fisher Scientific, catalog number: 50-230-8606 )
- Novagen rLysozyme (EMD Millipore, catalog number: 71110-4 )
- Novagen Benzonase (EMD Millipore, catalog number: 70664-3 )
- PMSF (Sigma-Aldrich, catalog number: 78830-5G )
- Aprotinin (Sigma-Aldrich, catalog number: A1153-5MG )
- Roche Complete Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 04-693-124-001 )
- Takara Chaperone plasmid set (Takara Bio Company, catalog number: 3340 )
- Amylose resin (New England Biolabs, catalog number: E8021S )
- Nitrocellulose membrane (Bio-Rad Laboratories, catalog number: 162-0115 )
- PVDF (EMD Millipore, catalog number: IPVH00010 )
- I-BLOCK (Life Technologies, Invitrogen™, catalog number: T2015 )
- Tween-20 (Sigma-Aldrich, catalog number: P9416 )
- Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
- Sodium azide (Sigma-Aldrich, catalog number: S2002 )
- Glycine (Sigma-Aldrich, catalog number: G8898 )
- Tris (Sigma-Aldrich, catalog number: T1503 )
- Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
- AminoLink Immobilization kit (Pierce Antibodies, catalog number: 44894 )
- Hydrochloric acid (HCl) (Thermo Fisher Scientific, catalog number: A144-212 )
- Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
- Ethanol by Acros(Thermo Fisher Scientific, catalog number: 61509-0020 )
- Methanol (Thermo Fisher Scientific, catalog number: BP1105-4 )
- Na2HPO4
- KH2PO4
- KCl
- NaCl
- CNBr-activated sepharose (Sigma-Aldrich, catalog number: C9142 )
- 3x PBST (see Recipes)
- Glycine elution buffer (see Recipes)
- Low pH wash buffer (see Recipes)
- High pH wash buffer (see Recipes)
- Coupling buffer (see Recipes)
Equipment
- Nickel-chromatography column (Thermo Fisher Scientific, catalog number: 50-230-8606 )
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Wirschell, M. and Porter, M. E. (2013). Generation of Polyclonal Specific Antibodies. Bio-protocol 3(11): e779. DOI: 10.21769/BioProtoc.779.
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分类
免疫学 > 抗体分析 > 抗体-抗原相互作用
生物化学 > 蛋白质 > 分离和纯化
生物化学 > 蛋白质 > 免疫检测
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