Detection of HBV C Protein Phosphorylation in the Cell
细胞中的HBV C蛋白磷酸化位点检测
发布: 2015年08月05日第5卷第15期 DOI: 10.21769/BioProtoc.1551 浏览次数: 7941
评审: Smita NairVarpu MarjomakiAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2014
Abstract
Among the seven serines and one threonine in the carboxyl-terminus of HBV C protein, all but one (serine 183) appear in the context of RxxS/T consensus phosphoacceptor motifs and also overlap with other consensus motifs, such as S/TP, RS, SPRRR, RRRS/T, or RRxS/T, suggesting that various cellular kinases phosphorylate these residues. To determine whether threonine and/or serine (serines 157, 164, 170, 172, 178, and 180, and threonine 162, adw subtype) of HBV C protein are indeed phosphoacceptor residues in cells, Huh7 were transfected with a series of C-protein-expressing mutants, labeled with 32P-orthophosphate for 14 h, and then lysed. The 32Pi-labeled lysates were immunoprecipitated with anti-HBc antibody, and the 32Pi-labeled immunoprecipitated C proteins were detected by autoradiography.
Materials and Reagents
- Huh7 hepatoma cells (Japanese Collection of Research Bioresources Cell Bank, catalog number: JCRB0403 )
- Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Gibco®, catalog number: 12800-017 )
- Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 16000-044 )
- Penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
- Plasmid; HBV WT C protein (STSSSS) in plasmid HBV-P-def of pcDNA3 backbone, phosphoacceptor-site mutants (ATAAAA, AAAAAA, SSSSSS, and ASAAAA) in plasmid HBV-P-def in pcDNA3 backbone, pcDNA3-GFP
- Polyethylenimine (Polysciences, catalog number: 23966 )
- Opti-MEM (Life Technologies, Gibco®, catalog number: 31985-062 )
- OptiMEM (Life Technologies, Gibco®, catalog number: 31985-070 )
- Dialyzed FBS (Life Technologies, Gibco®, catalog number: 26400044 )
- 1 mCi orthophosphate [32Pi] (PerkinElmer Inc., catalog number: NEX053 )
- Polyclonal rabbit anti-HBc antibody (home-made, Jung et al., 2012)
- Protein A/G Plus agarose beads (Calbiochem®, catalog number: IP05 )
- Tris-HCl (pH 8.5) (Sigma-Aldrich, catalog number: T6066 )
- EDTA (Sigma-Aldrich, catalog number: E5134 )
- Nonidet P-40 (Sigma-Aldrich, catalog number: CA630 )
- NaF (Sigma-Aldrich, catalog number: 201154 )
- β-glycerophosphate (USB, catalog number: 155-56-2 )
- Sodium orthovanadate (Sigma-Aldrich, catalog number: s6508 )
- Protease inhibitors (Calbiochem®, catalog number: 535142 )
- Tris-HCl (pH 6.8) (Sigma-Aldrich, catalog number: T6066)
- SDS (Sigma-Aldrich, catalog number: L-3771 )
- β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
- Bromophenol blue (Sigma-Aldrich, catalog number: 114391 )
- Glycerol (USB, catalog number: 16374 )
- Lysis buffer (see Recipes)
- 2x sample buffer (see Recipes)
Equipment
- 10-cm dishes (Corning Incorporated, catalog number: 430167 )
- PVDF membranes (Bio-Rad Laboratories, AbD Serotec®, catalog number: 162-0177 )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Jung, J. and Kim, K. (2015). Detection of HBV C Protein Phosphorylation in the Cell. Bio-protocol 5(15): e1551. DOI: 10.21769/BioProtoc.1551.
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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 > 免疫检测
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