Separation and Detection of Phosphorylated and Nonphosphorylated BvgA, a Bordetella pertussis Response Regulator, in vivo and in vitro
体内和体外分离和检测一种百日咳杆菌反应调节蛋白BvgA的磷酸化和非磷酸化形式
发布: 2013年11月20日第3卷第22期 DOI: 10.21769/BioProtoc.970 浏览次数: 11708
评审: Fanglian He
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Apr 2013
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Abstract
Protein phosphorylation plays a central role in signal transduction in bacteria. However, separation and detection of the phosphorylated protein from its nonphosphorylated form remain challenging. Here we describe a method to detect phosphorylation of the Bordetella pertussis response regulator BvgA, which is phosphorylated at an aspartate residue (Boulanger et al., 2013). This method is based on the proprietary adduct, Phos-tagTM, a dinuclear metal complex, which together with Zn2+ or Mn2+, forms a complex with a phosphomonoesterdianion, such as the phosphorylated aspartate of a response regulator (Barbieri and Stock, 2008; Kinoshita and Kinoshita-Kikuta, 2011). For in vivo detection, B. pertussis cells are lysed in mild formic acid at 4 °C to minimize the disruption of the phospho-aspartate bond, and the phosphorylated BvgA is separated from its nonphosphorylated form by electrophoresis (SDS-PAGE) containing Phos-tagTM. Both forms of BvgA are subsequently detected by Western Blot analysis. Quantification of the level of phosphorylated BvgA formed after treatment with acetyl phosphate in vitro is also easily accomplished. Thus, this technique allows one to readily assess the levels of BvgA phosphorylation in B. pertussis and in E. coli under different laboratory conditions in vivo or after phosphorylation under varying reaction conditions in vitro (this research was supported in part by the Intramural Research Program of the NIH, NIDDK).
Phosphorylation detection (磷酸化检测)
Response regulator (反应器)
Materials and Reagents
- Bordetella pertussis strain BP536 is the tet-resistant derivative of the clinical isolate Tohama I strain (laboratory inventory)
- Formic acid (> 95%) (Sigma-Aldrich, catalog number: F0507 )
- NaOH (Thermo Fisher Scientific, catalog number: SS255-1 )
- Tris base (MP Biomedicals, catalog number: 819620 )
- Bromophenol blue (Sigma-Aldrich, catalog number: B8026 )
- Glycerol (Invitrogen, catalog number: 15514-011 )
- Butanol (Sigma-Aldrich, catalog number: 15467-9 )
- 30% 29:1 Acrylamide/bis-acrylamide (Sigma-Aldrich, catalog number: A3574 )
- 10% SDS (Hoefer, catalog number: GR155-1 )
- Phos-tagTM acrylamide (Wako Pure Chemical Industries, catalog number: AAL-107 )
- Zn(NO3)2 (Sigma-Aldrich, catalog number: 228737 )
- Ammonium persulfate (Sigma-Aldrich, catalog number: 3678 )
- N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
- 0.5 M EDTA, pH 8.0 (Research Genetics, catalog number: 750009 )
- MOPS (Fluka, catalog number: 69947 )
- Sodium metabisulfite (Na2S2O5) (Sigma-Aldrich, catalog number: S9000 )
- Glycine (Sigma-Aldrich, catalog number: G7126-1KG )
- Methanol (EMD Millipore, catalog number: MX0485-7 )
- PBS buffer (Gibco, catalog number: 2014-10 )
- BactoTM proteose peptone (Becton, Dickinson and Company, catalog number: 211684 )
- DifcoTM Bordet Gengou agar base (Becton, Dickinson and Company, catalog number: 248200 )
- Defibrinated sheep blood (LAMPIRE® Biological Labs, catalog number: 7239001 )
- Lithium potassium acetyl phosphate (Sigma-Aldrich, catalog number: A0262-500MG )
- Colloidal Blue staining kit (Life Technologies, catalog number: LC6025 )
- Tween-20 (Bio-Rad Laboratories, catalog number: M3524 )
- Non-fat dry milk (Giant Food Store, catalog number: 688267078330 )
- Monoclonal anti-BvgA antibody (laboratory inventory)
- HRP-conjugated Secondary Antibody (Santa Cruz, catalog number: SC2005 )
- Amersham ECL Primer Western Blotting Detection System (General Electric Company, catalog number: RPN2232 )
- PVDF filter (Invitrogen, catalog number: LC6025 )
- BSA (Albumin from bovine serum) (Sigma-Aldrich, catalog number: A2153 )
- 1 M formic acid (see Recipes)
- 5 N NaOH (see Recipes)
- 1 M Tris-Cl solutions (see Recipes)
- 1% Bromophenol blue (see Recipes)
- 5x Loading Solution (see Recipes)
- Water-saturated butanol (see Recipes)
- 5 mM Phos-tagTM acrylamide (see Recipes)
- 10 mM Zn(NO3)2 (see Recipes)
- 10% APS (see Recipes)
- 1x MOPS Running Buffer, pH 7.8 (see Recipes)
- Transfer Buffer (see Recipes)
- BG agar plates (see Recipes)
- 200 mM acetyl phosphate (see Recipes)
- 1% or 5% BSA in PBS (see Recipes)
- 0.05% Tween-20 in PBS (see Recipes)
- 1% Non-fat milk in PBS (see Recipes)
Equipment
- Polyester-tipped applicator (Puritan Medical, catalog number: 25-806 1PD )
- Spectrophotometer
- Mini gel cassettes (1.0 mm) (Invitrogen, catalog number: NC2010 )
- 10, 12 or 15-well combs (1.0 mm) (Invitrogen, catalog number: NC3015 )
- XCell SureLock® Mini-Cell (Invitrogen, catalog number: EI0001 )
- Platform shaker
- Eppendorf microfuge
- Bio-Rad Mini-Protean II (Bio-Rad Laboratories)
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Chen, Q., Boulanger, A., Hinton, D. M. and Stibitz, S. (2013). Separation and Detection of Phosphorylated and Nonphosphorylated BvgA, a Bordetella pertussis Response Regulator, in vivo and in vitro. Bio-protocol 3(22): e970. DOI: 10.21769/BioProtoc.970.
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分类
微生物学 > 微生物信号传导 > 磷酸化
细胞生物学 > 细胞信号传导 > 磷酸化
生物化学 > 蛋白质 > 修饰
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