Helicase Assays
解旋酶活性检测试验
发布: 2014年03月20日第4卷第6期 DOI: 10.21769/BioProtoc.1079 浏览次数: 15017
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2013
Abstract
Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair involve helicase activity. Here, we provide a protocol to analyze helicase activities in vitro. In this protocol, the DNA helicase protein Merkel cell polyomavirus large T-antigen was expressed in the mammalian cell line HEK293 and immoblized on an IgG resin. The helicase assay is performing while the protein is immoblized on IgG resin.
Keywords: Helicase assay (解旋酶活性测定)
Large T-antigen (大T抗原)
MCV (MCV)
Materials and Reagents
- HEK293
- Dulbecco’s modified eagle medium high glucose (DMEM) (Life Technologies, catalog number: 11965-084 )
- Fetal bovine serum (FBS) (Hyclone, catalog number: SH30071.03 )
- DPBS without Ca2+ and Mg2+ (Life Technologies, catalog number: 14190136 )
- Trypsin/EDTA (Life Technologies, catalog number: 25300054 )
- IgG sepharose6 fast flow (GE, catalog number : 17-0969-01 )
- M13mp18 DNA (New England Biolab, catalog number: N4040S )
- DNA oligo: CCAGGGTTTTCCCAGTCACGACGTTGTAAAC
- DNA polymerase I klenow fragment (New England Biolab, catalog number: M0210S )
- 100 mM dCTP (Promega Corporation, atalog number: U1221 )
- 100 mM dGTP (Promega Corporation, catalog number: U1211 )
- 100 mM dATP (Promega Corporation, catalog number: U1201 )
- 100 mM ATP (Roche Diagnostics, catalog number: 11140965001 )
- [gamma-32P] dATP
- DNA loading dye
- Polyacrylamide gel
- 1 M Tris (pH 7.6) (see Recipes)
- 0.1 M Tris (pH 7.6) (see Recipes)
- 5 M NaCl stock (see Recipes)
- 1 M MgCl2 stock (see Recipes)
- 10 mM MgCl2 (see Recipes)
- 0.1 M PMSF (Sigma-Aldrich, catalog number: 78830 ) (see Recipes)
- 0.1 M DTT (Sigma-Aldrich, catalog number: 43819 ) (see Recipes)
- 1 mg/ml Aprotinin (Roche Diagnostics, catalog number: 10236624001 ) (see Recipes)
- 1 mg/ml Leupeptin (Roche Diagnostics, catalog number: 11017101001 ) (see Recipes)
- 1 mg/ml Pepstatin A (Roche Diagnostics, catalog number: 10253286001 ) (see Recipes)
- 10% NP40 stock (see Recipes)
- 1% Bovine serum albumin (BSA) (Sigma-Aldrich) (see Recipes)
- 10 mg/ml BSA (see Recipes)
- 10x TBE buffer (see Recipes)
- IPP400 buffer (see Recipes)
- Helicase assay buffer (see Recipes)
- Stop buffer (see Recipes)
- Acrylamide gel (see Recipes)
Equipment
- 20 G needle (BD, catalog number: 305175 )
- PowerPac basic power supply (Bio-Rad Laboratories, catalog number: 164-5050 )
- Mini-Protean tetra cell (Bio-Rad Laboratories, catalog number: 165-8000 )
- Sorvall RC 6 plus centrifuge and Sorvall SA-600 rotor (Thermo Fisher Scientific)
- 37 °C, 5% CO2 cell culture incubator
- Typhoon 9410 variable mode Imager
- Storage phosphor screens and cassettes (GE)
- Fisher Vortex Genie 2 (Thermo Fisher Scientific, catalog number: 12-812 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Wang, X., Li, J., Diaz, J. and You, J. (2014). Helicase Assays. Bio-protocol 4(6): e1079. DOI: 10.21769/BioProtoc.1079.
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分类
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 蛋白质 > 活性
生物化学 > 蛋白质 > 表达
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