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Crude Preparation of Lipopolysaccharide from Helicobacter pylori for Silver Staining and Western Blot
粗制备幽门螺杆菌脂多糖用于银染和蛋白质印迹   

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Abstract

This protocol provides an easy and rapid method to prepare lipopolysaccharide from the gastric pathogen Helicobacter pylori for visualization on acrylamide gels by silver staining and for detecting the presence of Lewis antigens by Western blot. The silver staining is a four-step procedure, involving a 20 min-oxidation step, a 10 min-silver staining step, a 2-10 min color development step and finally a 1-min color termination step. Lipopolysaccharide from H. pylori wild-type and corresponding mutants analyzed by this method are described in a recent publication (Li et al., 2017). This crude preparation of LPS for silver staining is also applicable in other Gram-negative bacteria.

Keywords: Lipopolysaccharide(脂多糖), Crude preparation(粗制备), Silver staining(银染), Western blot(蛋白质印迹), Helicobacter pylori(幽门螺杆菌)

Background

Lipopolysaccharide (LPS) is a large and variable complex glycolipid that makes up the outer leaflet of the outer membranes of most Gram-negative bacteria. It is typically composed of three domains: a hydrophobic domain termed lipid A (or endotoxin), which is embedded in the outer membrane; a relatively conserved non-repeating core-oligosaccharide; and a variable O-antigen, which extends from the cell to the external environment. A unique feature of H. pylori lipopolysaccharide O-antigen is the presence of fucosylated oligosaccharide structures that mimic human Lewis antigens. Large-scale extraction of highly pure LPS from Gram-negative bacteria is labor-intensive and time-consuming. Here, in this protocol, we describe in detail the use of an easy and rapid crude preparation of LPS from the gastric pathogen Helicobacter pylori for visualization by silver staining and Lewis antigen expression by Western blot.

Materials and Reagents

  1. Pipette tips
  2. Inoculating loops (10 μl) (Copan Diagnostics, catalog number: 8177CS20H )
  3. Aluminum foil
  4. PVDF membrane (0.2 μm) (Merck, catalog number: ISEQ00010 )
  5. H. pylori cells
  6. Columbia blood agar (CBA) plates (Autobio Diagnostics, catalog number: M0109-2 )
  7. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 746398-500G )
  8. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 746436-500G )
  9. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 795410-500G )
  10. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: 795496-500G )
  11. NaOH (Sigma-Aldrich, catalog number: S5881-500G )
  12. Proteinase K (Sigma-Aldrich, catalog number: P8044-1G )
  13. Ethanol (Sigma-Aldrich, catalog number: 24102-5L-R )
  14. SDS (Sigma-Aldrich, catalog number: L3771-1KG )
  15. Glycerol (Sigma-Aldrich, catalog number: G9012-1L )
  16. Tris base (Sigma-Aldrich, Roche Diagnostics, catalog number: 11814273001 )
  17. Glycine (Sigma-Aldrich, catalog number: G8898-1KG )
  18. Bromphenol blue (AMRESCO, catalog number: 0449-50G )
  19. β-Mercaptoethanol (AMRESCO, catalog number: 0482-100ML )
  20. Periodic acid (Sigma-Aldrich, catalog number: P7875 )
  21. Acetic acid (BDH, catalog number: 100015N )
  22. Ammonium persulfate (Sigma-Aldrich, catalog number: A3678 )
  23. Ammonium hydroxide (Sigma-Aldrich, catalog number: 320145 )
  24. Silver nitrate (Sigma-Aldrich, catalog number: 209139 )
  25. Citric acid (Sigma-Aldrich, catalog number: C7129 )
  26. Formalin (37% formaldehyde) (Sigma-Aldrich, catalog number: 252549 )
  27. Freshly-made 15% SDS-PAGE gels
  28. 30% Acrylamide/Bis (Bio-Rad Laboratories, catalog number: 1610157 )
  29. TEMED (Bio-Rad Laboratories, catalog number: 1610801 )
  30. Methanol (Sigma-Aldrich, catalog number: 34860-4X4L-R )
  31. Bovine serum albumin (BSA) (AMRESCO, catalog number: 0332-100G )
  32. Tween-20 (Solarbio, catalog number: T8220-500 ml )
  33. Mouse anti-Lex (Santa Cruz Biotechnology, catalog number: sc-59471 )
  34. Mouse anti-Ley (Santa Cruz Biotechnology, catalog number: sc-59472 )
  35. Mouse anti-Lea (Santa Cruz Biotechnology, catalog number: sc-51512 )
  36. Mouse anti-Leb (Santa Cruz Biotechnology, catalog number: sc-51513 )
  37. Secondary rabbit anti-mouse peroxidase-conjugated IgM antibody (Jackson ImmunoResearch Laboratories, catalog number: 315-035-049 )
  38. Chemiluminescent peroxidase substrate-1 (Sigma-Aldrich, catalog number: CPS1120 )
  39. PBS (pH 7.2) (10x) (see Recipes)
  40. SDS-PAGE running buffer (10x, see Recipes)
  41. 0.1 N NaOH solution (see Recipes)
  42. LPS lysis buffer (see Recipes)
  43. Oxidation solution (see Recipes)
  44. Silver staining solution (see Recipes)
  45. Color developer solution (see Recipes)
  46. Termination solution (see Recipes)
  47. SDS-PAGE transfer buffer (10x, see Recipes)
  48. TBS (10x, see Recipes)

Equipment

  1. Pipettes (Eppendorf, catalog numbers: 4920000024 , 4920000059 , 4920000067 , 4920000083 )
  2. Centrifuges (Eppendorf, catalog number: 5418 R )
  3. Glass wares
  4. Water bath
  5. Rotary shaker
  6. Cell electrophoresis tank (Bio-Rad Laboratories, catalog number: 1658001 )
  7. Electrophoresis power supply (Bio-Rad Laboratories, catalog number: 1645070 )
  8. Semi-dry electrophoretic transfer system (Bio-Rad Laboratories, catalog number: 1703940 )
  9. pH meter
  10. Spectrophotometer (Shimadzu, model: UV-1601 UV-Visible)
  11. Digital camera
  12. Luminescent Image Analyzer (Fujifilm, model: LAS-3000 )

Software

  1. Image reader LAS 3000 V2.2

Procedure

  1. LPS sample preparation
    1. Culture H. pylori strains on CBA plates, incubate at 37 °C for 24 h under microaerobic conditions.
    2. Use a loop (10 μl) to harvest H. pylori cells grown on CBA plates (24 h growth) into 1 ml of cold 1x PBS (see Recipes) to a turbidity of 3.0 at OD600.
    3. Centrifuge the H. pylori cells at 5,000 x g for 5 min, discard supernatant.
    4. Resuspend the pellet in 100 µl LPS lysis buffer (see Recipes) and heat the sample at 100 °C for 10 min, and allow to cool at room temperature.
    5. Add 5 μl Proteinase K (20 mg/ml) to the cooled samples, and incubate in a 55 °C water bath overnight.
    6. Load 10 μl of the above-obtained LPS sample per well to the 15% SDS-PAGE gel and electrophorese with 1x SDS-PAGE running buffer (see Recipes).
    7. Once the gel electrophoresis is complete, remove the gel from the apparatus and trim away the stacking gel.

  2. LPS silver staining
    1. Rinse the gel 3 times with ddH2O.
    2. Transfer the gel to a separate ware with freshly-made oxidation solution (see Recipes), oxidize for 20 min on a rotary shaker (70 rpm/min) (all shaking in this protocol is done at room temperature).
    3. Transfer the gel to a separate clean ware, rinse and wash with ddH2O for 3 times, each time with 200 ml ddH2O on a rotary shaker for 10 min.
    4. Transfer the gel to a separate ware with freshly-made silver staining solution (see Recipes), stain for 10 min on a rotary shaker.
      Note: The silver staining solution must be freshly-made, which can be made during the final wash of step B4. During the staining process, the ware should be wrapped in aluminum foil, but make sure the foil is not in contact with the solution.
    5. Transfer the gel to a separate clean ware, rinse and wash for 3 times, each time with 200 ml ddH2O on a rotary shaker for 10 min.
    6. Transfer the gel to a separate ware with freshly-made color developer solution (see Recipes), develop for 2-10 min on a rotary shaker.
      Note: The color developer solution must be freshly-made, which can be made during the final wash of step B6. The color will develop on the gel within to 2-10 min, the longer the developer solution remains in contact with the gel the darker the bands will become. Therefore, it is important to watch the gel develop. The operator must decide when the gel is sufficiently developed and transfer the gel to the termination solution (see Recipes).
    7. Transfer the gels to a separate ware with freshly-made termination solution for 1 min. The gels are then rinsed and stored in ddH2O.
    8. Use a digital camera to take images of the silver-stained LPS gels.

  3. Western blot for the detection of Lewis antigen expression
    Proceed from step 7 of Procedure A.
    1. Transfer LPS from the gel to PVDF membrane with 1x SDS-PAGE transfer buffer (see Recipes).
    2. Once the transfer is complete, remove the membrane from the apparatus.
    3. Rinse the membrane 3 times with ddH2O.
    4. Block the membrane with TBST (see Recipes) containing 3% BSA, on a rotary shaker for 2 h or overnight at 4 °C.
    5. Pour off the blocking solution.
    6. Probe with primary antibodies: mouse anti-Lex/y or anti-Lea/b (1:1,500) (Santa Cruz) in TBST containing 1% BSA, on a rotary shaker for 2 h or overnight at 4 °C.
    7. Pour off the primary antibody solution.
    8. Rinse and wash with TBST for 3 times, each time on a rotary shaker for 10 min.
    9. Probe with secondary rabbit anti-mouse peroxidase-conjugated IgM antibody (1:10,000) (Jackson ImmunoResearch Laboratories) in TBST, on a rotary shaker for 1 h.
    10. Pour off the secondary antibody solution.
    11. Rinse and wash with TBST for 3 times, each time on a rotary shaker for 10 min.
    12. Place the membrane on a flat sheet of plastic film.
    13. Add approximately 5 ml of the chemiluminescent peroxidase substrate-1 working solution (Sigma-Aldrich) on the membrane.
    14. Cover the membrane with a second flat sheet of plastic film.
    15. Detect chemiluminescence using a LAS-3000 Intelligent DarkBox (Fujifilm) (software Image reader LAS 3000 V2.2).

Data analysis

Figure 1 below illustrates LPS prepared from H. pylori wild-type strains 26695, P1, B128 and J99 analyzed by silver staining and Western blot using anti-Lewis X (Lex) and anti-Lewis Y (Ley).

  1. Silver staining: the fast migration bands stained black represent molecules comprising lipid A and core-oligosaccharide (Lipid A-core); the slower migration bands represent O-antigen. It is obvious that LPS preparations from the four H. pylori strains show a different staining pattern, indicating the strains carrying different LPS structures (Figure 1A).
  2. Western blot: LPS from H. pylori strains 26695, P1 and J99 were detected expressing both Lex and Ley antigens (Figure 1B, Lanes 1, 2 and 4), whereas LPS prepared from strain B128 was not detected expressing Lex/y (Figure 1B, Lane 3).


    Figure 1. Crude preparation of LPS from H. pylori strains for silver staining and Western blot

Recipes

Note: ddH2O is used for preparing all the solutions unless otherwise indicated.

  1. PBS (pH 7.2) (10x) (1 L)
    80 g NaCl
    2 g KCl
    14.4 g Na2HPO4
    2.4 g KH2PO4
    Add 900 ml H2O to dissolve all the reagents and adjust the pH to 7.2, then add H2O to make the total volume up to 1 L. The buffer is sterilized by autoclaving and stored at room temperature. The 1x working solution is prepared by mixing 100 ml 10x PBS buffer with 900 ml H2O
  2. 0.1 N NaOH (1 L)
    4 g NaOH
    The total volume is made up to 1 L with H2O. The buffer was sterilized by autoclaving and stored at room temperature
  3. LPS lysis buffer (20 ml)
    2 ml 20% SDS
    800 μl β-mercaptoethanol
    200 μl bromophenol blue
    2 ml glycerol
    15 ml 1 M Tris-HCl (pH 6.8)
  4. Oxidation solution (100 ml)
    0.7 g periodic acid
    40 ml EtOH
    5 ml acetic acid
    55 ml H2O
  5. Silver staining solution (150 ml)
    28 ml 0.1 N NaOH
    2 ml ammonium hydroxide (29.4%)
    115 ml H2O
    5 ml 20% silver nitrate (w/v)
    Note: This solution is mixed in the following order by adding 28 ml 0.1 N NaOH, 2 ml concentrated ammonium hydroxide (29.4%), and 115 ml H2O in a dedicated ware, then transfer the ware on the rotary shaker (50 rpm/min). Finally, add the 5 ml 20% silver nitrate dropwise to the solution, waiting for the brown precipitate to dissolve before adding more of silver nitrate solution. If the brown precipitate fails to dissolve, discard the solution and remake the solution by adding the 20% silver nitrate more slowly.
  6. Color developer solution (200 ml)
    Dissolve 1 g citric acid into 20 ml H2O, resulting into 5% citric acid:
    200 μl 5% citric acid
    100 μl 37% formaldehyde
    200 ml H2O
  7. Termination solution (100 ml)
    10 ml acetic acid
    90 ml ddH2O
  8. SDS-PAGE running buffer (10x) (1 L)
    30.3 g Tris base
    144 g glycine
    10 g SDS
    The total volume is made up to 1 L with H2O. The buffer was sterilized by autoclaving and stored at room temperature. The 1x working solution is prepared by mixing 100 ml 10x running buffer with 900 ml H2O
  9. SDS-PAGE transfer buffer (10x) (1 L)
    30.3 g Tris base
    144 g glycine
    The total volume is made up to 1 L with H2O. The buffer was sterilized by autoclaving and stored at room temperature. The 1x working solution is prepared by mixing 100 ml 10x transfer buffer with 700 ml H2O and 200 ml methanol
  10. TBS (10x) (1 L)
    80 g NaCl
    2 g KCl
    30 g Tris base
    Add 900 ml H2O to dissolve all the reagents and adjust the pH to be 7.4, then add H2O to make the total volume up to 1 L. The stocking buffer is sterilized by autoclaving and stored at room temperature. TBST is 1x TBS containing 0.1% Tween-20 (v/v)

Acknowledgments

This protocol described here and in Li et al. (2017) was adapted from protocols described in Apicella et al. (2008) and in Tsai et al. (1982). This work was supported by a grant from West China Hospital, Sichuan University entitled ‘1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University’ (ZY2016201).

References

  1. Apicella, M. A. (2008). Isolation and characterization of lipopolysaccharides. Methods Mol Biol 431: 3-13.
  2. Tsai, C. M. and Frasch, C. E. (1982). A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119(1): 115-119.

简介

该方案提供了一种从胃病原幽门螺杆菌制备脂多糖的简便快速方法,用于通过银染显示丙烯酰胺凝胶,并通过Western印迹检测Lewis抗原的存在。 银染是四步法,包括20分钟氧化步骤,10分钟银染色步骤,2-10分钟显色步骤,最后是1分钟颜色终止步骤。 来自H的脂多糖。 在最近的出版物(Li等人,2017)中描述了通过该方法分析的野生型和相应的突变体的幽门螺杆菌。 这种用于银染的LPS的粗制剂也适用于其他革兰氏阴性细菌。
【背景】脂多糖(LPS)是一种大而可变的复合糖脂,其组成了大多数革兰氏阴性细菌外膜的外叶。 它通常由三个结构域组成:称为脂质A(或内毒素)的疏水结构域,其嵌入外膜中; 相对保守的非重复核 - 低聚糖; 和从细胞延伸到外部环境的可变O-抗原。 H的独特功能。 幽门螺杆菌脂多糖O抗原是模拟人Lewis抗原的岩藻糖基化寡糖结构的存在。 从革兰氏阴性细菌大规模提取高纯度的LPS是劳动密集型和耗时的。 在这里,在这个协议中,我们详细地描述了使用来自胃病原幽门螺杆菌的容易和快速的粗制备制剂用于通过银染和Lewis抗原通过蛋白质印迹进行表达。

关键字:脂多糖, 粗制备, 银染, 蛋白质印迹, 幽门螺杆菌

材料和试剂

  1. 移液器提示
  2. 接种环(10μl)(Copan Diagnostics,目录号:8177CS20H)
  3. 铝箔
  4. PVDF膜(0.2μm)(Merck,目录号:ISEQ00010)
  5. 小时。幽门螺杆菌细胞
  6. 哥伦比亚血琼脂(CBA)板(Autobio Diagnostics,目录号:M0109-2)
  7. 氯化钠(NaCl)(Sigma-Aldrich,目录号:746398-500G)
  8. 氯化钾(KCl)(Sigma-Aldrich,目录号:746436-500G)
  9. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:795410-500G)
  10. 磷酸氢二钾(K 2/2 HPO 4)(Sigma-Aldrich,目录号:795496-500G)
  11. NaOH(Sigma-Aldrich,目录号:S5881-500G)
  12. 蛋白酶K(Sigma-Aldrich,目录号:P8044-1G)
  13. 乙醇(Sigma-Aldrich,目录号:24102-5L-R)
  14. SDS(Sigma-Aldrich,目录号:L3771-1KG)
  15. 甘油(Sigma-Aldrich,目录号:G9012-1L)
  16. Tris碱(Sigma-Aldrich,Roche Diagnostics,目录号:11814273001)
  17. 甘氨酸(Sigma-Aldrich,目录号:G8898-1KG)
  18. 溴酚蓝(AMRESCO,目录号:0449-50G)
  19. β-巯基乙醇(AMRESCO,目录号:0482-100ML)
  20. 周期酸(Sigma-Aldrich,目录号:P7875)
  21. 乙酸(BDH,目录号:100015N)
  22. 过硫酸铵(Sigma-Aldrich,目录号:A3678)
  23. 氢氧化铵(Sigma-Aldrich,目录号:320145)
  24. 硝酸银(Sigma-Aldrich,目录号:209139)
  25. 柠檬酸(Sigma-Aldrich,目录号:C7129)
  26. 福尔马林(37%甲醛)(Sigma-Aldrich,目录号:252549)
  27. 新鲜15%SDS-PAGE凝胶
  28. 30%丙烯酰胺/双(Bio-Rad Laboratories,目录号:1610157)
  29. TEMED(Bio-Rad Laboratories,目录号:1610801)
  30. 甲醇(Sigma-Aldrich,目录号:34860-4X4L-R)
  31. 牛血清白蛋白(BSA)(AMRESCO,目录号:0332-100G)
  32. 吐温-20(Solarbio,目录号:T8220-500ml)
  33. 小鼠抗Le x (Santa Cruz Biotechnology,目录号:sc-59471)
  34. 小鼠抗Le y (Santa Cruz Biotechnology,目录号:sc-59472)
  35. 小鼠抗Le a (Santa Cruz Biotechnology,目录号:sc-51512)
  36. 小鼠抗Le b (Santa Cruz Biotechnology,目录号:sc-51513)
  37. 次级兔抗小鼠过氧化物酶缀合的IgM抗体(Jackson ImmunoResearch Laboratories,目录号:315-035-049)
  38. 化学发光过氧化物酶底物-1(Sigma-Aldrich,目录号:CPS1120)
  39. PBS(pH 7.2)(10x)(参见食谱)
  40. SDS-PAGE运行缓冲液(10x,参见食谱)
  41. 0.1 N NaOH溶液(参见食谱)
  42. LPS裂解缓冲液(参见食谱)
  43. 氧化溶液(见配方)
  44. 银染溶液(参见食谱)
  45. 彩色显影剂溶液(参见食谱)
  46. 终止解决方案(见配方)
  47. SDS-PAGE转移缓冲液(10x,参见食谱)
  48. TBS(10x,见配方)

设备

  1. 移液器(Eppendorf,目录号:4920000024,4920000059,4920000067,4920000083)
  2. 离心机(Eppendorf,目录号:5418 R)
  3. 玻璃器皿
  4. 水浴
  5. 旋转振动筛
  6. 细胞电泳槽(Bio-Rad Laboratories,目录号:1658001)
  7. 电泳电源(Bio-Rad Laboratories,目录号:1645070)
  8. 半干电泳转移系统(Bio-Rad Laboratories,目录号:1703940)
  9. pH计
  10. 分光光度计(Shimadzu,型号:UV-1601 UV-Visible)
  11. 数码相机
  12. 发光图像分析仪(Fujifilm,型号:LAS-3000)

软件

  1. 图像读取器LAS 3000 V2.2

程序

  1. LPS样品制备
    1. 文化H。幽门螺杆菌菌株在CBA平板上,在微量条件下于37℃孵育24小时。
    2. 使用一个环(10μl)收获H。幽门螺杆菌细胞在CBA平板上生长(24小时生长)到1ml冷的1x PBS(参见食谱)中,OD 600处的浊度为3.0。
    3. 离心H。幽门螺杆菌细胞在5,000×g下5分钟,弃去上清液。
    4. 将沉淀重悬于100μlLPS裂解缓冲液(参见食谱),并将样品在100℃下加热10分钟,并使其在室温下冷却。
    5. 向冷却的样品中加入5μl蛋白酶K(20 mg / ml),并在55°C水浴中孵育过夜。
    6. 将10μl上述获得的LPS样品/孔加入到15%SDS-PAGE凝胶中,并用1x SDS-PAGE运行缓冲液电泳(参见食谱)。
    7. 一旦凝胶电泳完成,从设备中取出凝胶,并剪掉堆积凝胶。

  2. LPS银染
    1. 用ddH 2 O冲洗凝胶3次。
    2. 将凝胶转移到具有新鲜氧化溶液的另外的器皿(参见食谱),在旋转振荡器上(70rpm / min)氧化20分钟(在本方案中均匀振荡在室温下进行)。
    3. 将凝胶转移到单独的清洁剂中,用ddH 2 O 3冲洗并洗涤3次,每次在旋转振荡器上用200ml ddH 2 O处理10分钟。
    4. 将凝胶转移到具有新鲜银染色溶液的单独的器皿(参见食谱),在旋转振荡器上染色10分钟。
      注意:银染溶液必须是新鲜的,可以在步骤B4的最后洗涤过程中进行。在染色过程中,器皿应用铝箔包裹,但要确保箔片不与溶液接触。
    5. 将凝胶转移到单独的清洁剂中,冲洗并洗涤3次,每次在旋转振荡器上用200ml ddH 2 O进行10分钟。
    6. 将凝胶转移到具有新鲜显色剂溶液的独立制品(参见食谱)中,在旋转振荡器上培养2-10分钟。
      注意:显色剂溶液必须是新鲜的,可以在步骤B6的最后洗涤过程中进行。颜色将在凝胶上发展至2-10分钟,显影剂溶液保持与凝胶接触的时间越长,条带将变得越暗。因此,重要的是观察凝胶的发展。操作者必须决定凝胶是否充分发展,并将凝胶转移到终止溶液中(参见食谱)。
    7. 将凝胶转移到具有新鲜终止溶液的单独零件1分钟。然后将凝胶漂洗并储存在ddH 2 O中。
    8. 使用数码相机拍摄银色LPS凝胶的图像。

  3. Western blot检测Lewis抗原表达
    从步骤A的步骤7开始。
    1. 使用1x SDS-PAGE转移缓冲液将LPS从凝胶转移到PVDF膜(参见食谱)
    2. 转移完成后,从设备中取出膜。
    3. 用ddH 2 O冲洗膜3次。
    4. 将TBST(见食谱)含有3%BSA,在旋转振荡器上封闭2小时或4℃过夜。
    5. 倾倒阻塞解决方案。
    6. 用一级抗体进行检测:小鼠抗Le 或 或 a / b (1: 1,500)(Santa Cruz)在含有1%BSA的TBST中,在旋转振荡器上搅拌2小时或在4℃下过夜。
    7. 倒出一级抗体溶液
    8. 用TBST冲洗和洗涤3次,每次旋转摇床10分钟。
    9. 在TBST中用二级兔抗小鼠过氧化物酶缀合的IgM抗体(1:10,000)(Jackson ImmunoResearch Laboratories)探针,在旋转振荡器上1小时。
    10. 倒出二抗溶液。
    11. 用TBST冲洗和洗涤3次,每次旋转摇床10分钟。
    12. 将膜放在一块塑料薄膜上。
    13. 在膜上加入约5ml的化学发光过氧化物酶底物-1工作溶液(Sigma-Aldrich)
    14. 用第二张塑料薄膜覆盖膜。
    15. 使用LAS-3000智能DarkBox(Fujifilm)(软件图像读取器LAS 3000 V2.2)检测化学发光。

数据分析

图1示出了通过银染色和Western印迹分析的幽门螺杆菌野生型菌株26695,P1,B128和J99制备的LPS,使用抗Lewis X(Le < )和抗Lewis Y(Le y )。

  1. 银染色:染色黑色的快速迁移带表示包含脂质A和核 - 低聚糖(脂质A核)的分子;较慢的迁移带代表O-抗原。显然,来自四个H的LPS制剂。幽门螺杆菌菌株显示不同的染色模式,表明携带不同LPS结构的菌株(图1A)
  2. 蛋白质印迹:来自H的LPS。检测到幽门螺杆菌菌株26695,P1和J99,表达Le x 和Le y 抗原(图1B,泳道1,2和4),而LPS制备来自菌株B128未检测到表达Le x / y (图1B,泳道3)。


    图1.来自H的LPS的粗制备。幽门螺杆菌菌株用于银染和Western印迹

食谱

注意:除非另有说明,否则O用于准备所有解决方案。

  1. PBS(pH 7.2)(10x)(1L)
    80克NaCl
    2克KCl
    14.4g Na 2 HPO 4
    2.4g KH PO 4
    加入900ml H 2 O以溶解所有试剂,并将pH调节至7.2,然后加入H 2 O使总体积达到1L。缓冲液为通过高压灭菌消毒并在室温下储存。 1x工作溶液通过将100ml 10×PBS缓冲液与900ml H 2 O 2 /
  2. 0.1N NaOH(1L)
    4g NaOH
    总体积为H 2 O 2的1L。缓冲液通过高压灭菌消毒并在室温下储存
  3. LPS裂解缓冲液(20ml) 2 ml 20%SDS
    800μlβ-巯基乙醇
    200μl溴酚蓝
    2毫升甘油
    15 ml 1M Tris-HCl(pH 6.8)
  4. 氧化溶液(100ml)
    0.7克周期酸
    40 ml EtOH
    5 ml乙酸
    55ml H 2 O O
  5. 银染溶液(150 ml)
    28 ml 0.1 N NaOH 2ml氢氧化铵(29.4%)
    115ml H 2 O ○
    5毫升20%硝酸银(w / v)
    注意:通过加入28ml 0.1N NaOH,2ml浓氢氧化铵(29.4%)和115ml H 2 O 2,将该溶液按以下顺序混合。 在专门的零件中,然后将转盘上的零件(50 rpm / min)转移。最后,将5ml 20%硝酸银滴加到溶液中,等待棕色沉淀物溶解,然后再加入更多的硝酸银溶液。如果棕色沉淀物不能溶解,请丢弃溶液,并通过加入20%硝酸银更缓慢地重新溶解。
  6. 显色剂溶液(200毫升)
    将1g柠檬酸溶解于20ml H 2 O中,得到5%柠檬酸:
    200微升5%柠檬酸
    100μl37%甲醛
    200ml H 2 O O
  7. 终止溶液(100ml)
    10 ml醋酸 90毫升ddH 2 O O
  8. SDS-PAGE运行缓冲液(10x)(1L)
    30.3克Tris碱基
    144克甘氨酸
    10g SDS
    总体积为H 2 O 2的1L。缓冲液通过高压灭菌消毒并在室温下储存。 1x工作溶液通过将100ml 10x运行缓冲液与900ml H 2 O 2 /
  9. SDS-PAGE转移缓冲液(10x)(1L)
    30.3克Tris碱基
    144克甘氨酸
    总体积为H 2 O 2的1L。缓冲液通过高压灭菌消毒并在室温下储存。 1x工作溶液通过将100ml 10x转移缓冲液与700ml H 2 O 2和200ml甲醇混合而制备,
  10. TBS(10x)(1 L)
    80克NaCl
    2克KCl
    30克Tris碱基
    加入900ml H 2 O以溶解所有试剂并调节pH至7.4,然后加入H 2 O使总体积达到1L。放养缓冲液通过高压灭菌消毒并在室温下储存。 TBST是含有0.1%Tween-20(v / v)
    的1x TBS

致谢

这里描述的和在Li等人(2017)中描述的这种协议是根据在Apicella等人(2008)和蔡氏等人(1982)。这项工作得到了四川大学华西医院资助,题为“四川大学华西医院”1.3.5“优秀学科项目”(ZY2016201)。

参考

  1. Apicella,M.A。(2008)。 脂多糖的分离和表征。方法Mol Biol 431 :3-13。
  2. Tsai,C.M.和Frasch,C.E。(1982)。 用于检测聚丙烯酰胺凝胶中脂多糖的敏感银染色。 Anal Biochem < 119(1):115-119。
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引用:Li, H. and Benghezal, M. (2017). Crude Preparation of Lipopolysaccharide from Helicobacter pylori for Silver Staining and Western Blot. Bio-protocol 7(20): e2585. DOI: 10.21769/BioProtoc.2585.
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