Immunoprecipitation of Proteins in Caenorhabditis elegans
免疫沉淀法检测秀丽隐杆线虫蛋白质
(*contributed equally to this work) 发布: 2015年04月05日第5卷第7期 DOI: 10.21769/BioProtoc.1436 浏览次数: 17785
评审: Peichuan ZhangAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2014
Abstract
Immunoprecipitation (IP) is a biochemical technique to precipitate a protein out of solution using an antigen that can specifically bind to that protein. IP can be performed to isolate and concentrate one particular protein from a sample of thousands of different proteins. IP is also readily performed to pull down interacting proteins of complexes out of solution. This protocol outlines the methods used to IP proteins in whole worm lysates and their preparation for detection on Western blots using denaturing conditions.
Materials and Reagents
- Transgenic Caenohabditis elegans (C. elegans) strain with over-expressed protein of interest
Note: Depending on protein expression and specificity of antibody, this protocol can also be used to immunoprecipitate endogenous proteins.
- OP50 Escherichia coli (E. coli) (C. elegans Genetic Center)
- Trizma hydrochloride (Sigma-Aldrich, catalog number: T5941 )
- Trizma base (Sigma-Aldrich, catalog number: T1503 )
- Tris base (BioShop, catalog number: TRS001 )
- Bacto-tryptone (BD, catalog number: 211705 )
- NaCl (BioShop, catalog number: SOD002 )
- Cholesterol (95%) (Sigma-Aldrich, catalog number: C8503 )
- Agar (Sigma-Aldrich, catalog number: A1296 )
- Na2HPO4 (Sigma-Aldrich, catalog number: S0876 )
- KH2PO4 (Sigma-Aldrich, catalog number: P0662 )
- MgSO4·7H2O (BioShop, catalog number: MAG521 )
- EDTA (BioBasic, catalog number: EB0185 )
- NP-40 (BioShop, catalog number: NON.505 )
- PMSF (Roche, catalog number: 10837091001 )
- Na3VO4 (Sigma-Aldrich, catalog number: S6508 )
- Pepstatin-A (Sigma-Aldrich, catalog number: P4265 )
- NaF (Sigma-Aldrich, catalog number: S7920 )
- cOmplete protease inhibitor cocktail tablets (Roche, catalog number: 4693159001 )
- SDS (Caledon, catalog number: 7771 )
- Glycerol (BioShop, catalog number: GLY001 )
- β-mercaptoethanol (Sigma-Aldrich, catalog number: M7154 )
- Bromophenol blue (Sigma-Aldrich, catalog number: B5525 )
- BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23225 )
- Siliconized low-binding tips 200-1,000 μl (Denville Scientific Inc., catalog number: P3193-S )
- Siliconized low-binding tips 200 μl (Denville Scientific Inc., catalog number: P3010-S )
- Protein A/G PLUS-Agarose non-conjugated beads (Santa Cruz Biotechnology, catalog number: SC-2003 )
- EZview Red ANTI-FLAG M2 Affinity Gel conjugated beads (Sigma-Aldrich, catalog number: F2426 )
- GFP polyclonal antibody, rabbit (Genscript, catalog number: A01388 )
- GFP monoclonal antibody (B-2) (mouse) (Santa Cruz Biotechnology, catalog number: SC-9996 )
- Donkey anti-rabbit IgG-HRP (Santa Cruz Biotechnology, catalog number: SC-2313 )
- Goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, catalog number: SC-2005 )
- MYOB dry mix (see Recipes)
- MYOB plates (see Recipes)
- M9 buffer (see Recipes)
- Lysis buffer (see Recipes)
- SDS-PAGE sample buffer (see Recipes)
Equipment
- Refrigerated microcentrifuge (Eppendorf, model: 5417R )
- Large centrifuge (Eppendorf, model: 5810R )
- Rotisserie agitator (Barnstead Thermolyne LabQuake, model: C415110 )
- Sonicator (Branson Sonifier, model: 450 )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Chan, K. K., Seetharaman, A., Selman, G. and Roy, P. J. (2015). Immunoprecipitation of Proteins in Caenorhabditis elegans. Bio-protocol 5(7): e1436. DOI: 10.21769/BioProtoc.1436.
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分类
生物化学 > 蛋白质 > 免疫检测
生物化学 > 蛋白质 > 免疫检测
生物化学 > 蛋白质 > 分离和纯化
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