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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   

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参见作者原研究论文

本实验方案简略版
Molecular Microbiology
Apr 2013

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).

Keywords: Phos-tag (磷标记), Phosphorylation detection (磷酸化检测), Response regulator (反应器)

Materials and Reagents

  1. Bordetella pertussis strain BP536 is the tet-resistant derivative of the clinical isolate Tohama I strain (laboratory inventory)
  2. Formic acid (> 95%) (Sigma-Aldrich, catalog number: F0507 )
  3. NaOH (Thermo Fisher Scientific, catalog number: SS255-1 )
  4. Tris base (MP Biomedicals, catalog number: 819620 )
  5. Bromophenol blue (Sigma-Aldrich, catalog number: B8026 )
  6. Glycerol (Invitrogen, catalog number: 15514-011 )
  7. Butanol (Sigma-Aldrich, catalog number: 15467-9 )
  8. 30% 29:1 Acrylamide/bis-acrylamide (Sigma-Aldrich, catalog number: A3574 )
  9. 10% SDS (Hoefer, catalog number: GR155-1 )
  10. Phos-tagTM acrylamide (Wako Pure Chemical Industries, catalog number: AAL-107 )
  11. Zn(NO3)2 (Sigma-Aldrich, catalog number: 228737 )
  12. Ammonium persulfate (Sigma-Aldrich, catalog number: 3678 )
  13. N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
  14. 0.5 M EDTA, pH 8.0 (Research Genetics, catalog number: 750009 )
  15. MOPS (Fluka, catalog number: 69947 )
  16. Sodium metabisulfite (Na2S2O5) (Sigma-Aldrich, catalog number: S9000 )
  17. Glycine (Sigma-Aldrich, catalog number: G7126-1KG )
  18. Methanol (EMD Millipore, catalog number: MX0485-7 )
  19. PBS buffer (Gibco, catalog number: 2014-10 )
  20. BactoTM proteose peptone (Becton, Dickinson and Company, catalog number: 211684 )
  21. DifcoTM Bordet Gengou agar base (Becton, Dickinson and Company, catalog number: 248200 )
  22. Defibrinated sheep blood (LAMPIRE® Biological Labs, catalog number: 7239001 )
  23. Lithium potassium acetyl phosphate (Sigma-Aldrich, catalog number: A0262-500MG )
  24. Colloidal Blue staining kit (Life Technologies, catalog number: LC6025 )
  25. Tween-20 (Bio-Rad Laboratories, catalog number: M3524 )
  26. Non-fat dry milk (Giant Food Store, catalog number: 688267078330 )
  27. Monoclonal anti-BvgA antibody (laboratory inventory)
  28. HRP-conjugated Secondary Antibody (Santa Cruz, catalog number: SC2005 )
  29. Amersham ECL Primer Western Blotting Detection System (General Electric Company, catalog number: RPN2232 )
  30. PVDF filter (Invitrogen, catalog number: LC6025 )
  31. BSA (Albumin from bovine serum) (Sigma-Aldrich, catalog number: A2153 )
  32. 1 M formic acid (see Recipes)
  33. 5 N NaOH (see Recipes)
  34. 1 M Tris-Cl solutions (see Recipes)
  35. 1% Bromophenol blue (see Recipes)
  36. 5x Loading Solution (see Recipes)
  37. Water-saturated butanol (see Recipes)
  38. 5 mM Phos-tagTM acrylamide (see Recipes)
  39. 10 mM Zn(NO3)2 (see Recipes)
  40. 10% APS (see Recipes)
  41. 1x MOPS Running Buffer, pH 7.8 (see Recipes)
  42. Transfer Buffer (see Recipes)
  43. BG agar plates (see Recipes)
  44. 200 mM acetyl phosphate (see Recipes)
  45. 1% or 5% BSA in PBS (see Recipes)
  46. 0.05% Tween-20 in PBS (see Recipes)
  47. 1% Non-fat milk in PBS (see Recipes)

Equipment

  1. Polyester-tipped applicator (Puritan Medical, catalog number: 25-806 1PD )
  2. Spectrophotometer
  3. Mini gel cassettes (1.0 mm) (Invitrogen, catalog number: NC2010 )
  4. 10, 12 or 15-well combs (1.0 mm) (Invitrogen, catalog number: NC3015 )
  5. XCell SureLock® Mini-Cell (Invitrogen, catalog number: EI0001 )
  6. Platform shaker
  7. Eppendorf microfuge
  8. Bio-Rad Mini-Protean II (Bio-Rad Laboratories)

Procedure

  1. Whole cell lysate preparation for in vivo detection of BvgA phosphorylation
    Note: This protocol has been successfully used with another gram-negative bacteria, E. coli.
    1. To collect bacteria sample, Bordetella pertussis strain BP536, grown at 37 °C for 2 days on BG agar plate, is swabbed from the plate with a polyester-tipped applicator and resuspended in 1.5 ml of PBS.
    2. An aliquot from step A1 is used to determine the OD600 reading.
    3. A 0.3 ml aliquot from step A1 is centrifuged at 15,600 x g in Eppendorf microfuge for 1 min at room temperature. The supernatant is removed, and the resulting pellet is frozen in dry ice. It can be used directly or stored at -80 °C.
    4. The frozen pellet is treated as follows.
      Note: Volumes are based on an OD600 reading of 0.5 and a pellet obtained from 0.3 ml, one should adjust the volumes proportionally based on the determined OD600.
      First, 33 μl of ice-cold 1 M formic acid is added, and the pellet is disrupted by pipetting repeatedly. Immediately, a freshly made ice-cold solution (27 μl) containing 2 μl of 5 N NaOH (to neutralize the acid), 10 μl H2O, and 15 μl of 5x Loading Solution is added.
      Note: The color of the solution turns yellow due to the acid, but the bromophenol blue changes back to blue once it enters the gel.
    5. 4 μl of the resulting cell lysate are loaded onto the Phos-tagTM gel for electrophoresis.
      Note: Do not boil the treated cell lysate; the lysate should be kept on ice before loading.

  2. Phos-tagTM SDS-PAGE gel electrophoresis (Recipe makes one mini gel.)
    4% stacking gel

    30% Acrylamide/bis-acrylamide
    417.5 μl
    1 M Tris pH 6.8
    1,093 μl
    10% SDS
    31.3 μl
    10% APS (freshly prepared)
    25 μl
    TEMED
    5 μl
    H2O
    1,570 μl


    10% resolving gel

    30% Acrylamide/bis-acrylamide
    2,085 μl
    1 M Tris pH 6.8
    2,185 μl
    10% SDS
    62.5 μl
    5 mM Phos-tagTM
    93.8 μl
    10 mM Zn(NO3)2
    93.8 μl
    10% APS (freshly prepared)
    25 μl
    TEMED
    5 μl
    H2O
    1,715 μl

    1. For resolving gel: Acrylamide, Tris, SDS, Phos-tagTM, Zn(NO3)2, and H2O are mixed. 10% APS and TEMED are added, and the solution is immediately poured into the gel cassette. The solution is then overlaid with 1 ml of water-saturated butanol. The resolving gel polymerizes in ~40 min at room temperature.
    2. The butanol is poured off of the polymerized resolving gel, and the gel surface is then washed with water. Excess water is removed using absorbent paper.
    3. For stacking gel: Acrylamide, Tris, SDS, and H2O are mixed. 10% APS and TEMED are added, and the solution is immediately poured into the gel cassette. The comb is quickly inserted (avoiding the introduction of air bubbles) and the stacking gel is allowed to polymerize for 20 min at room temperature.
    4. After insertion of the gel cassette into the electrophoresis apparatus, the ice-cold 1x MOPS Running Buffer is added. The entire apparatus is then placed into an ice-filled bucket and the bucket is placed on a platform shaker so that it can be gently rotated until the temperature of the Running Buffer in the chamber has cooled to 4 °C. This typically takes at least 30 min.
      Note: Place only one gel cassette in one XCell SureLock® Mini-Cell to ensure that the temperature remains cold during electrophoresis.
    5. The shaker is turned off and the samples are loaded using a pipetman.
    6. After resumption of gentle shaking, electrophoresis is performed with constant 100 V for 30 min, 120 V for 30 min, and then 150 V until the bromophenol blue dye is ~ 1 cm from the bottom of the gel. (Temperature within the inner gel chamber will rise to 10 °C to 15 °C.)
    7. For Western blot analysis, the gel is washed with 100 ml Transfer Buffer containing 1 mM EDTA for 10 min to remove Zn2+ from the gel, followed by washing again with 100 ml Transfer Buffer for 20 min.
    8. The gel is transferred to a PVDF filter, which was previously rinsed with methanol and then with Transfer Buffer, in a Bio-Rad Mini-Protean II apparatus filled with ice-cold Transfer Buffer at 100 constant voltage for 1 h at 4 °C.
    9. The antibody detection of BvgA is carried out at room temperature as follows: After the transfer, the PVDF filter is blocked by washing with 5% BSA in PBS for 1 h, and then incubated with monoclonal anti-BvgA antibody (1:5,000) in 1% BSA in PBS for 1 h. Three 10-min washes with 0.05% Tween-20 in PBS are conducted before incubation with HRP-conjugated Secondary Antibody (1:2,000) in 1% non-fat milk in PBS for 1 h. The PVDF filter is washed three times for 10 min, each, with 0.05% Tween-20 in PBS, and then developed with Amersham ECL Primer Western Blotting Detection System, according to the manufacture’s instruction.

  3. Separation of in vitro phosphorylated purified protein
    1. To phosphorylate in vitro, 9 μl purified BvgA (from ~1 to 25 pmol) is mixed with 1 μl 200 mM acetyl phosphate on ice and then incubated for the desired amount of time at 37 °C.
    2. Reaction is stopped by placing the sample on dry ice.
    3. Sample is mixed with 2.5 μl 5x Loading Solution (final concentration of 1x), separated on PhostagTM acrylamide gel, washed, and detected by Western analysis as described above.
    4. Alternately, gel can be stained. In this case, the gel is first washed 3 times in 100 ml of water for 10 min. The gel is then treated with Colloidal Blue following manufacturer’s instructions.
      Note: For best results, use either Colloidal Blue or classic Coomassie blue staining rather than other types of protein stains.
      Note: The optimal level of purified protein for loading is ~ 1 pmol for Western analysis and ~25 pmol for gel staining


      Figure 1. Separation of phosphorylated BvgA from unphosphorylated BvgA by Phos-tagTM SDS-PAGE, analyzed by Western blot. First lane shows the in vivo lysate of B. pertussis, which contains both phosphorylated BvgA (BvgA~P) and nonphosphorylated BvgA (BvgA). Lanes 2 and 3 show purified BvgA, which has been phosphorylated in vitro by the addition of acetyl phosphate or incubated in the absence of acetyl phosphate, respectively.

Recipes

  1. 1 M formic acid
    5 ml of formic acid (> 95%) in 95 ml of H2O
    Stored at 4 °C
  2. 5 N NaOH
    5 ml 10 N NaOH
    5 ml H2O
    Stored at 4 °C
  3. 1 M Tris-Cl solutions (for 1 L)
    121.14 g Tris base adjusted to pH 6.8 or to pH 8.0 with HCl at 4 °C
    Filter sterilized
  4. 1% Bromophenol blue
    1 g bromophenol blue in 100 ml H2O
  5. 5x Loading Solution
    1% SDS
    65 mM Tris-Cl (pH 6.8)
    25% glycerol
    0.02% bromophenol blue
    Stored at 4 °C
  6. Water-saturated butanol
    4 ml water added to 40 ml butanol
    Mix by shaking
    Butanol will be the top layer
  7. 5 mM Phos-tagTM acrylamide
    10 mg Phos-tagTM acrylamide dissolved in 100 μl methanol
    Solution is pipetted repeatedly to dissolve the acrylamide
    Add 3.2 ml H2O
    Solution is centrifuged at 2,000 x g for 10 min to remove white precipitate
    Stored at 4 °C in dark
  8. 10 mM Zn(NO3)2
    2.97 g Zn(NO3)2 dissolved in 1 L H2O
    Filter sterilized
  9. 10% APS
    1 g ammonium persulfate dissolved in 10 ml H2O
    Solution is freshly prepared
  10. 1x MOPS Running Buffer, pH 7.8
    100 mM Tris-CL, pH 7.8
    100 mM MOPS, pH 7.8
    0.1% SDS
    5 mM sodium metabisulfite
    For 1 L
    12.1 g Tris base
    20.9 g MOPS
    10 ml 10% SDS
    0.95 g sodium metabisulfite (Na2S2O5)
    Solution is adjusted to pH 7.8 with 10 N HCl at 4 °C
    Stored at 4 °C
  11. Transfer Buffer
    3 g Tris base
    14.4 g glycine
    200 ml of methanol
    800 ml of H2O
  12. BG agar plates
    10 g DifcoTM Bordet Gengou agar base
    3.3 g BactoTM proteose peptone
    3.3 ml glycerol added to 350 ml H2O
    The mixture is autoclaved
    Cooled to 46 °C before adding 50 ml defibrinated sheep blood
    Pour warm solution into petri dishes and allow to cool to room temperature.
    Stored at 4 °C
  13. 200 mM acetyl phosphate
    0.0039 g lithium potassium acetyl phosphate dissolved in 105.7 μl 20 mM Tris-Cl, pH 8 Solution is freshly made
    Stored on ice
  14. 1% or 5% BSA in PBS
    1 g or 5 g BSA
    100 ml PBS
  15. 0.05% Tween-20 in PBS
    0.05 ml of Tween-20
    100 ml of PBS
  16. 1% non-fat milk in PBS
    1 g non-fat dry milk
    100 ml of PBS

Acknowledgments

This protocol is based on the previously published paper Boulanger et al. (2013). The research was supported in part by the Intramural Research Program of the NIH, NIDDK.

References

  1. Barbieri, C. M. and Stock, A. M. (2008). Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag-based reagents. Anal Biochem 376(1): 73-82.
  2. Boulanger, A., Chen, Q., Hinton, D. M. and Stibitz, S. (2013). In vivo phosphorylation dynamics of the Bordetella pertussis virulence-controlling response regulator BvgA. Mol Microbiol 88(1): 156-172.
  3. Kinoshita, E. and Kinoshita-Kikuta, E. (2011). Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling. Proteomics 11(2): 319-323.

简介

蛋白质磷酸化在细菌的信号转导中起着中心作用。然而,从其非磷酸化形式分离和检测磷酸化蛋白仍然是挑战性的。在这里我们描述了检测百日咳博德特氏菌响应调节剂BvgA的磷酸化的方法,其在天冬氨酸残基被磷酸化(Boulanger等人,2013)。该方法基于专有的加合物Phos-tag TM sup/TM,其是双核金属络​​合物,其与Zn 2+或Mn 2+反应, ,与磷酸二酯酶形成复合物,例如应答调节剂的磷酸化天冬氨酸(Barbieri和Stock,2008; Kinoshita和Kinoshita-Kikuta,2011)。对于体内检测,在4℃下在轻度甲酸中裂解百日咳细胞以使磷酸 - 天冬氨酸键的破坏最小化,并且通过包含Phos标签的电泳(SDS-PAGE)将磷酸化的BvgA从其非磷酸化形式分离> TM 。随后通过蛋白质印迹分析检测两种形式的BvgA。还容易实现在体外用乙酰磷酸盐处理后形成的磷酸化BvgA的水平的量化。因此,该技术允许容易地评估B中BvgA磷酸化的水平。百日咳和 。大肠杆菌在不同实验室条件下在体内或在不同反应条件下在体外磷酸化后(本研究部分由NIH的Intramural Research Programme支持, NIDDK)。

关键字:磷标记, 磷酸化检测, 反应器

材料和试剂

  1. 百日咳博德特氏菌菌株BP536是临床分离株Tohama I菌株(实验室库存)的抗tet抗性衍生物
  2. 甲酸(> 95%)(Sigma-Aldrich,目录号:F0507)
  3. NaOH(Thermo Fisher Scientific,目录号:SS255-1)
  4. Tris碱(MP Biomedicals,目录号:819620)
  5. 溴酚蓝(Sigma-Aldrich,目录号:B8026)
  6. 甘油(Invitrogen,目录号:15514-011)
  7. 丁醇(Sigma-Aldrich,目录号:15467-9)
  8. 30%29:1丙烯酰胺/双丙烯酰胺(Sigma-Aldrich,目录号:A3574)
  9. 10%SDS(Hoefer,目录号:GR155-1)
  10. Phos-tag TM TM丙烯酰胺(Wako Pure Chemical Industries,目录号:AAL-107)
  11. Zn(NO 3)2(Sigma-Aldrich,目录号:228737)
  12. 过硫酸铵(Sigma-Aldrich,目录号:3678)
  13. N,N,N',N'-四甲基乙二胺(TEMED)(Sigma-Aldrich,目录号:T9281)
  14. 0.5M EDTA,pH8.0(Research Genetics,目录号:750009)
  15. MOPS(Fluka,目录号:69947)
  16. 偏亚硫酸氢钠(Na 2 SS 2 O 5)(Sigma-Aldrich,目录号:S9000)
  17. 甘氨酸(Sigma-Aldrich,目录号:G7126-1KG)
  18. 甲醇(EMD Millipore,目录号:MX0485-7)
  19. PBS缓冲液(Gibco,目录号:2014-10)
  20. Bacto TM 蛋白胨(Becton,Dickinson and Company,目录号:211684)
  21. Difco TM Bordet Gengou琼脂基料(Becton,Dickinson and Company,目录号:248200)
  22. 去纤维蛋白绵羊血(LAMPIRE Biological Labs,目录号:7239001)
  23. 乙酰磷酸锂钾(Sigma-Aldrich,目录号:A0262-500MG)
  24. 胶体蓝染色试剂盒(Life Technologies,目录号:LC6025)
  25. Tween-20(Bio-Rad Laboratories,目录号:M3524)
  26. 脱脂奶粉(Giant Food Store,目录号:688267078330)
  27. 单克隆抗BvgA抗体(实验室库存)
  28. HRP-偶联的二抗(Santa Cruz,目录号:SC2005)
  29. Amersham ECL Primer Western Blotting Detection System(General Electric Company,目录号:RPN2232)
  30. PVDF过滤器(Invitrogen,目录号:LC6025)
  31. BSA(来自牛血清的白蛋白)(Sigma-Aldrich,目录号:A2153)
  32. 1 M甲酸(参见配方)
  33. 5 N NaOH(见配方)
  34. 1 M Tris-Cl溶液(参见配方)
  35. 1%溴酚蓝(见配方)
  36. 5x加载解决方案(参见配方)
  37. 水饱和的丁醇(见配方)
  38. 5mM Phos-tag TM 丙烯酰胺(参见配方)
  39. 10mM Zn(NO 3)2(参见配方)
  40. 10%APS(参见配方)
  41. 1x MOPS运行缓冲液,pH 7.8(参见配方)
  42. 传输缓冲区(参见配方)
  43. BG琼脂平板(见配方)
  44. 200 mM乙酰磷酸(见配方)
  45. 1%或5%BSA的PBS溶液(参见配方)
  46. 0.05%Tween-20的PBS溶液(参见配方)
  47. 1%PBS中的脱脂牛奶(参见配方)

设备

  1. 聚酯涂抹器(Puritan Medical,目录号:25-806 1PD)
  2. 分光光度计
  3. 微型凝胶盒(1.0mm)(Invitrogen,目录号:NC2010)
  4. 10,12或15-孔梳(1.0mm)(Invitrogen,目录号:NC3015)
  5. XCell SureLock Mini-Cell(Invitrogen,目录号:EI0001)
  6. 平台振动器
  7. Eppendorf微量离心器
  8. Bio-Rad Mini-Protean II(Bio-Rad Laboratories)

程序

  1. 用于体内的全细胞裂解物制备检测BvgA磷酸化
    注意:此协议已成功用于另一种革兰氏阴性菌E.coli。
    1. 为了收集细菌样品,将在BG琼脂板上在37℃下生长2天的百日咳博德特氏菌菌株BP536用聚酯头涂布器从板上擦拭并重悬于1.5ml PBS中。 br />
    2. 使用来自步骤A1的等分试样来测定OD <600>读数
    3. 将来自步骤A1的0.3ml等分试样在15,600×g下在Eppendorf微量离心机中在室温下离心1分钟。除去上清液,将所得沉淀物在干冰中冷冻。它可以直接使用或储存在-80°C
    4. 如下处理冷冻的沉淀。
      注意:体积基于0.5的OD <600>读数和从0.3ml获得的沉淀,应根据测定的OD 600来按比例调节体积。
      首先,加入33μl冰冷的1M甲酸,并通过重复吹打破碎沉淀。立即加入含有2μl5N NaOH(以中和酸),10μlH 2 O和15μl5x上样溶液的新鲜制备的冰冷溶液(27μl)。
      注意:溶液的颜色由于酸而变黄,但是溴酚蓝一旦进入凝胶就变回蓝色。
    5. 将4μl所得细胞裂解物加载到用于电泳的Phos-tag TM 凝胶上。
      注意:不要煮沸处理的细胞裂解物;裂解物应在装载前保存在冰上。

  2. Phos-tag TM SDS-PAGE凝胶电泳(Recipe make one mini gel。)
    4%堆积凝胶

    30%丙烯酰胺/双丙烯酰胺 417.5微升
    1M Tris pH 6.8
    1,093微升
    10%SDS
    31.3微升
    10%APS(新鲜制备)
    25μl
    TEMED
    5微升
    H sub 2 O
    1,570μl


    10%解决凝胶

    30%丙烯酰胺/双丙烯酰胺 2,085μl
    1M Tris pH 6.8
    2,185微升
    10%SDS
    62.5微升
    5mM Phos-tag TM
    93.8μl
    10mM Zn(NO 3)2 sub。 93.8μl
    10%APS(新鲜制备)
    25μl
    TEMED
    5微升
    H sub 2 O
    1,715微升

    1. 对于拆分凝胶:丙烯酰胺,Tris,SDS,Phos-tag TM,Zn(NO 3)2和H 2 SO 4,/sub> O混合。 加入10%APS和TEMED,并将溶液立即倒入凝胶盒中。 然后用1ml水饱和的丁醇覆盖溶液。 拆分凝胶在室温下〜40分钟内聚合
    2. 从聚合的拆分凝胶中倒出丁醇,然后用水洗涤凝胶表面。 用吸水纸除去多余的水。
    3. 对于堆叠胶:将丙烯酰胺,Tris,SDS和H 2 O混合。 加入10%APS和TEMED,并将溶液立即倒入凝胶盒中。 梳子迅速插入(避免 引入气泡),并使堆叠的凝胶在室温下聚合20分钟
    4. 在将凝胶盒插入电泳装置后,加入冰冷的1x MOPS运行缓冲液。然后将整个设备放入充满冰的桶中,将桶放在平台振荡器上,使其可以轻轻旋转,直到室中运行缓冲液的温度冷却至4℃。这通常需要至少30分钟。
      注意:在一个XCell SureLock ® Mini-Cell中只放置一个凝胶盒,以确保电泳期间温度保持冷。
    5. 关闭振动器,使用移液管加载样品。
    6. 在温和振荡恢复后,以恒定的100V持续30分钟,120V持续30分钟,然后150V直到溴酚蓝染料距离凝胶底部〜1cm为止进行电泳。 (内部凝胶室内的温度将升至10℃至15℃)
    7. 对于蛋白质印迹分析,用含有1mM EDTA的100ml转移缓冲液洗涤凝胶10分钟以从凝胶中除去Zn 2+,随后再次用100ml转移缓冲液洗涤20分钟。
    8. 将凝胶转移到PVDF过滤器中,该PVDF过滤器预先用甲醇然后用转移缓冲液在装有冰冷转移缓冲液的Bio-Rad Mini-Protean II装置中在100℃恒压下在4℃漂洗1小时。
    9. BvgA的抗体检测在室温下如下进行:转移后,通过用PBS中的5%BSA洗涤1小时来封闭PVDF过滤器,然后与单克隆抗BvgA抗体(1:5000)一起温育1%BSA的PBS溶液1小时。在与HRP缀合的二抗(1:2000)在PBS中的1%脱脂奶中孵育1小时之前,用在PBS中的0.05%Tween-20进行三次10分钟洗涤。将PVDF过滤器洗涤三次,每次10分钟,用PBS中的0.05%Tween-20洗涤,然后根据制造商的说明用Amersham ECL Primer Western印迹检测系统显影。

  3. 体外分离磷酸化的纯化蛋白质
    1. 为了体外磷酸化,将9μl纯化的BvgA(约1至25pmol)与1μl200mM乙酰磷酸在冰上混合,然后在37℃下温育所需的时间。 br />
    2. 将样品置于干冰上停止反应。
    3. 样品与2.5μl5x上样溶液(最终浓度为1x)混合,在Phostag TM丙烯酰胺凝胶上分离,洗涤并如上所述通过Western分析检测。
    4. 或者,可以染色凝胶。在这种情况下,首先将凝胶在100ml水中洗涤3次,每次10分钟。然后根据制造商的说明书用胶体蓝处理凝胶 注意:为了获得最佳效果,使用胶体蓝或经典考马斯蓝染色,而不是其他类型的蛋白质染色。
      注意:对于Western分析,加载的纯化蛋白的最佳水平为〜1pmol,对于凝胶染色为〜25pmol。


      图1.通过Western印迹分析的通过Phos-标记的SDS-PAGE分离磷酸化的BvgA与未磷酸化的BvgA。第一泳道显示在体内 B的溶胞产物。百日咳,其含有磷酸化的BvgA(BvgA〜P)和非磷酸化的BvgA(BvgA)。泳道2和3显示纯化的BvgA,其已经通过添加乙酰磷酸酯或在不存在乙酰磷酸酯下分别孵育而在体外被磷酸化。

食谱

  1. 1 M甲酸
    在95ml H 2 O中的5ml甲酸(> 95%)
    储存在4°C
  2. 5 N NaOH
    5ml 10N NaOH
    5毫升H 2 O 2 / 储存在4°C
  3. 1 M Tris-Cl溶液(1 L)
    121.14g Tris碱,在4℃下用HCl调节至pH 6.8或pH 8.0 过滤灭菌
  4. 1%溴酚蓝
    1g在100ml H 2 O中的溴酚蓝
  5. 5x加载解决方案
    1%SDS
    65mM Tris-Cl(pH6.8) 25%甘油 0.02%溴酚蓝
    储存在4°C
  6. 水饱和的丁醇
    将4ml水加入到40ml丁醇中 摇动混合
    丁醇将是顶层
  7. 5mM Phos-标记丙烯酰胺 10mg溶于100μl甲醇中的Phos-tag TM丙烯酰胺 将溶液重复移液以溶解丙烯酰胺 加入3.2ml H 2 O 2 / 将溶液在2,000×g离心10分钟以除去白色沉淀物
    储存于4℃,黑暗
  8. 10mM Zn(NO 3)2 sub。 溶解在1L H 2 O中的2.97g Zn(NO 3)2溶液
    过滤灭菌
  9. 10%APS
    1g溶解在10ml H 2 O中的过硫酸铵 溶液是新鲜制备的
  10. 1x MOPS运行缓冲液,pH 7.8
    100mM Tris-CL,pH7.8
    100mM MOPS,pH 7.8 0.1%SDS
    5mM偏亚硫酸氢钠 对于1 L
    12.1克Tris碱
    20.9克MOPS
    10ml 10%SDS
    0.95克偏亚硫酸氢钠(Na 2 SS 2 O 5)
    用10N HCl在4℃下将溶液调节至pH 7.8 储存在4°C
  11. 传输缓冲区
    3克Tris碱
    14.4g甘氨酸
    200ml甲醇
    800ml H 2 O 2 /
  12. BG琼脂平板
    10 g Difco TM Bordet Gengou琼脂基料
    3.3g Bacto TM sup/TM蛋白胨
    将3.3ml甘油加入到350ml H 2 O中 将混合物高压灭菌
    冷却至46℃,然后加入50ml脱纤维绵羊血液
    将温热的溶液倒入陪替氏培养皿中,并允许冷却至室温。
    储存在4°C
  13. 200mM乙酰磷酸 0.0039g乙酰磷酸锂钾溶于105.7μl20mM Tris-Cl,pH8中的溶液新鲜制备溶液 存放在冰上
  14. 1%或5%BSA的PBS溶液
    1 g或5 g BSA
    100 ml PBS
  15. 0.05%Tween-20的PBS溶液中 0.05ml Tween-20 100ml的PBS
  16. 1%无脂牛奶的PBS溶液
    1克脱脂奶粉
    100ml的PBS

致谢

该协议基于先前发表的论文Boulanger等人(2013)。 该研究得到了NIH NIDDK的院内研究计划的部分支持。

参考文献

  1. Barbieri,C.M.and Stock,A.M。(2008)。 使用基于Phos标记的体外和体内监测反应调节剂天冬氨酸磷酸化的通用方法试剂。 Anal Biochem 376(1):73-82
  2. Boulanger,A.,Chen,Q.,Hinton,DM和Stibitz,S。(2013)。体内 磷酸化动力学百日咳博德特氏菌毒力控制反应调节剂BvgA。 Mol Microbiol 88(1):156-172。
  3. Kinoshita,E。和Kinoshita-Kikuta,E。(2011)。 在中性pH条件下改进的Phos-tag SDS-PAGE,用于高级蛋白磷酸化分析。 Proteomics 11(2):319-323
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
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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