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Western Blot Analysis of Chloroplast HSP70B in Chlorella Species
蛋白印迹分析小球藻物种中的叶绿体蛋白HSP70B   

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参见作者原研究论文

本实验方案简略版
Gene
Mar 2013

Abstract

Western blotting allows for the specific detection of proteins by an antibody of interest. This protocol utilizes isolation of total proteins protocol for Chlorella vulgaris prior to gel electrophoresis. After electrophoresis, the selected antibodies are used to detect and quantify levels of chloroplast HSP70B.

Materials and Reagents

  1. Species  
    Three Chlorella species were used: C. vulgaris, isolated from soil samples of Livingston Island, the South Shetland Archipelago, Antarctic; C. vulgaris strain 8/1, isolated in 1968 from thermal springs in the region of Rupite, Bulgaria, and cultivated in our laboratory since 1975 and Chlorella kesslery a mesophile, from the Trebon collection.
    Cultivation  
    Chlorella species were cultivated on TAP (Tris Acetate Phosphate) medium under continuous light of 60 μmol/m2/s and a temperature of 23 °C ± 0.1 °C in a Phytotron GC 400 growth chamber. The species were cultivated at this temperature because it is well known, that eurythermal algae, could be grown at a wide range of temperatures.
  2. Rabbit polyclonal antibody HSP70B cytoplasmic (Agrisera, catalog number: AS06 175 )
  3. Goat anti-rabbit IgG(H&l) HRP conjugated (Agrisera, catalog number: AS09 602 )
  4. Coomassie brilliant blue G 250
  5. Orthophosphoric acid (Valerus, catalog number: N 4420 )
  6. Trichloroacetic acid (TCA)
  7. Bovine serum albumin (BSA) (Applichem GmbH, catalog number: 1391 0025 )
  8. Albumin fraction V (pH 7.0)
  9. Medium Pure Nitrocellulose (NCM) (0.45 μm) (Bio-Rad Laboratories, catalog number: 162-0115 )
  10. Filter paper
  11. Sponge
  12. 4CN (4-chloro-naphthol) (Bio-Rad Laboratories, catalog number: N170-6535 )
  13. N,N′ N′ Tetramethylethylendiamine (TEMED) (Alfa Aesar, catalog number: N12536 )
  14. Laemmli sample buffer (see Recipes)
  15. Reagent of Bradford (see Recipes)
  16. 5x Laemmli buffer (see Recipes)
  17. Running buffer (see Recipes)
  18. Transfer buffer (see Recipes)
  19. SDS-PAGE gel (see Recipes)
  20. 30% Acrylamide/N,N’-methylenebisacrylamide (AA/MBA) (see Recipes)  
  21. 10% SDS (see Recipes)
  22. 10% Ammonium Persulfate (see Recipes)
  23. 1.5 M Tris HCl buffer (pH 8.8) (see Recipes)
  24. 1.0 M Tris HCl buffer (pH 6.8) (see Recipes)
  25. 4 M NaCl (see Recipes)
  26. 1.0 M Tris HCl buffer (pH 7.5) (see Recipes)
  27. 20% Tween 20 (see Recipes)
  28. Blocking buffer (see Recipes)
  29. Staining solution (see Recipes)
  30. 5% CH3COOH (see Recipes)
  31. Washing solution (see Recipes)
  32. 50 mM TBS-T buffer (see Recipes)
  33. HRP color development solution (see Recipes)

Equipment

  1. Motor
  2. Silica  quartz sand 0.6 mm (Valerus, catalog number: N 1760 )
  3. Centrifuge (Sigma-Aldrich, model: 1-15 K)
  4. Electrophoresis chamber Transfer unit Hoefer miniVE electrophotesis and electrotransfer unit (Hoefer, model: SE300-10A-1.0 )
  5. Mini Rocker Shaker MR-1

Software

  1. Image J program

Procedure

  1. Cells lysis
    1. Add 100 μl Lisys Solution (LS) to the pellet (Chankova et al., 2013b) transfer to a chilled mortar, add two spatulas of silica sand, grind in the mortar for 3 min, add 200 μl LS in the mortar to wash and transfer the material into an Eppendorf tube of 2 ml.
    2. Centrifuge material from step 1 for 10 min at 14,500 x g.
    3. Separate the supernatant and heat the supernatant for 5 min at t = 90 °С.
    4. Centrifuge for 5 min at 14,500 x g.
    5. Split the supernatant in 2 samples: The first one use for the determination of protein concentration; the second one keep at t = -20 °С.

  2. Determination of protein concentration (Bradford)
    1. Add 30 μl 20% TCA to 30 μl supernatant.
    2. Centrifuge for 5 min at 14,500 x g.
    3. Add 60 μl 0.1 N NaOH to the pellet and mix thoroughly. To obtain best result add twice 30 μl of 0.1 N NaOH.
    4. Take 14 μl, add 86 μl 0.15 М NaCl and 3 ml reagent of Bradford.
    5. Use calibration curve for quantity of protein (Table 1).
      For calibration curve:
      Stock solution – 0.5 mg/ml BSA
      Use Table 1 to determine every point of standard curve add 3 ml reagent of Bradford.

      Table 1. Calibration curve for quantity of protein

      N
      BSA (μg)
      BSA (0.5 mg/ml) vol (μl)
      NaCl (0.15 M) vol (μl)
      1
      0
      0
      100
      2
      5
      10
      90
      3
      10
      20
      80
      4
      15
      30
      70
      5
      20
      40
      60
      6
      25
      50
      50
      7
      30
      60
      40
      8
      35
      70
      30
      9
      40
      80
      20
      10
      45
      90
      10
      11
      50
      100
      0

  3. Protein electrophoresis
    1. Put about 100 ml of the 1x Laemmli buffer into cuvettes of electrophoresis module.
    2. Remove the comb and rinse the wells with buffer of SDS-PAGE gel.
    3. Pipet 10 μg protein into every well: adjust volumes so equal amount of protein is loaded (example: 10 μg protein are contained in 10 μl sample).
    4. Put the rest buffer in a bath of electrophoresis chamber (the volume must be always above minimum.).
    5. Run electrophoresis using the following parameters: 120 V and 16 mA for 3.5 h.
    6. When the electrophoresis is completed, remove the gel carefully.
      Note: The order of the dropping of the samples. Concentrated gel should be released.

  4. Transfer of proteins on the NCM
    1. Soak the gel for 15 min in buffer.
    2. Soak sponge and filter paper for sandwich in transfer buffer.
    3. Cut NCM. The size should be such as the size of the gel. Put NCM for 5 min in transfer buffer.
      Note: Mark the order of samples on the membrane! Label the membrane with a pencil.
    4. Make a sandwich.
      1. The stack is assembled on the black cathode side (see Figure 1):
        1. Center a packing sponge on the black cathode side.
        2. Center a packing sponge on the black catode (a).
        3. Lay one piece of wet filter paper on the sponge (b).
        4. Position the equilibrate gels on the filter paper(c).
        5. Lay the membrane on the gel (d).
        6. Lay one piece of wet filter paper on the membrane (e).
        7. Lay two packing sponges on the filter paper (f).
        8. A second transfer stack if added, is placed between these two sponges.


          Figure 1. Assembling a transfer stack (this is an original figure taken from the Technical Guide available at www. hoeferinc.com)

      2. Different parts of the sandwich press very well, to avoid bubbles.
      3. Different parts of the sandwich should be well moistened. You can "roll" them with a tube.
        1. Close the apparatus. Put in a chamber transfer buffer.
        2. Run blotting with the following parameters: 35 V and 250 mA for 2 h.

  5. Western blot
    1. After the transfer of proteins, place the membrane in blocking buffer at t = 4 °C. Incubate on a rocker platform for 1 h (following this step we have obtained the best results).
    2. Place gels in staining solution for 4-5 h.
    3. Wash for 3-4 h the gel with washing solution.
    4. Dilute primary antibody in blocking buffer (1:10,000) and incubate according to manufacturer’s instructions. Incubate on a rocker platform at t = 4 °C overnight.
    5. Wash the membrane in TBS-T buffer on a rocker platform in a following way: twice for 2 min (2 х 2 min), after that twice for 10 min (2 x 10 min).
    6. Prepare secondary antibody in blocking buffer (1:20,000) and incubate according to manufacturer’s instructions. Incubate on a rocker platform at RT for 2 h.
    7. Wash the membrane in TBS-T buffer on a rocker platform in a following way: Twice for 2 min (2 х 2 min), after that three times for 5 min (3 x 5 min).
    8. Visualize using HRP Color Development Solution, 4CN according manufacturer’s instructions.
    9. Scan the membrane. Calculate protein amount using Image J program.

    Recipes

    1. Laemmli sample buffer
      2% SDS
      5% 2-mercaptoehtanol
      10% glycerol
      0.002%(w/v) bromophenol blue
      62.5 mM Tris HCl (pH 6.8)
    2. Reagent of Bradford
      Dissolve 100 mg Coomassie brilliant blue G 250 and 50 ml 96% alcohol in a stirrer for 15 min. Add 94.5 ml 90% orthophosphoric acid.
      Add 900 ml deionized H2O and stir gently.
      Filtering through a folded filter paper and make up to 1 L with deionized water.
      Keep in a fridge at t = 4 °C.
    3. 5x Laemmli buffer
      15 g TRIS base
      72 g Glycine in 1 L deionized H2O
    4. Running Buffer
      Add 200 ml 5x Laemmli buffer + 10 ml 10% SDS to 1 L deionized H2O
    5. Transfer Buffer
      Add 200 ml 5x Laemmli buffer + 2 ml 10% SDS to + 200 ml ethanol to 1 L deionized H2O
    6. SDS-PAGE gel
      Note: Glass tiles should be cleared well with alcohol before preparing SDS-PAGE gel.
      1. Separating gel (12.5%) (Table 2)

        Table 2. Preparing of separating gel solution

        Number of mini-gels
        1
        2
        Deionized H2O
        3.2 ml
        6.4 ml
        Acrilamide/bisacrilamide (30%)
        4 ml
        8.0 ml
        1.5 M Tris HCl buffer, pH 8.8
        2.6 ml
        5.2 ml
        10% SDS
        100 μl
        200 μl
        10% APS
        100 μl
        200 μl
        TEMED
        10 μl
        20 μl

        1. Mix very carefully the components in a 50 ml Falcon tube to avoid bubbles.
        2. Insert separating gel between two glass plates of the chamber (about 1 cm below the boundary of tiles).
        3. Add deionized H2O carefully as a thin film using a syringe and wait about 15 min.
        4. Carefully remove the water; Wipe the water drops in the ends with filter paper.
      2. Stacking gel (Table 3)

        Table 3. Preparing of 4% stacking gel solution
        Number of mini-gels
        1
        2
        Deionized H2O
        1,370 μl
        2,740 μl
        Acrilamide/bisacrilamide (30%)
        330 μl
        660 μl
        1.0 M Tris HCl buffer, pH 6.8
        250 μl
        500 μl
        10% SDS
        20 μl
        40 μl
        10% APS
        20 μl
        40 μl
        TEMED
        2 μl
        4 μl

      3. Put the concentrated gel, insert the comb and wait until the gel polymerize.
      4. For an electrophoresis is better to prepare about 1,250 ml 1x Laemmli buffer. It can be used twice.
    7. 30% AA/MBA
      29.0 g + 1.0 g MBA dissolve in 72.5 ml deionized H2O, make up the volume to 100 ml, filter using 0.45 μm filter
      Keep at t = 4 °C less than 1 month.
    8. 10% SDS
      Dissolve 10 g SDS in 100 ml deionized H2O
    9. 10% Ammonium Persulfate
      Dissolve 1 g in 10 ml deionized H2O
      Keep at t = 4 °C less than 1 month.
    10. 1.5 M Tris HCl Buffer pH 8.8
      Dissolve 18.5 g Tris base in 80 ml deionized H2O, adjust to pH = 8.8 with concentrated HCl and make up the volume to 100 ml.
    11. 1.0 M Tris HCl Buffer pH 6.8
      Dissolve 12.114 g Tris base in 80 ml deionized H2O, adjust to pH= 6.8 with concentrated HCl and make up the volume to 100 ml.
    12. 50 mM TBS-T buffer
      1.0 M Tris HCl buffer (pH 7.5)
      200 mM NaCl
      0.1% Tween 20
    13. 4 M NaCl
      Dissolve 23.376 g NaCl in100 ml deionized H2O
    14. 1.0 M Tris HCl buffer (pH 7.5)
      Dissolve 12.114 g TRIS base in 80 ml deionized H2O, adjust to pH 7.5 with concentrated HCl and make up to the 100 ml.
    15. 20% Tween 20
      20 ml Tween make up to 100 ml deionized H2O.
    16. Blocking buffer
      Dissolve 5% fatless dry milk in 100 ml TBS-T buffer.
    17. Staining solution
      0.2% Coomassie Brilliant blue R- 250
      40% C2H5OH
    18. 5% CH3COOH
      Dissolve 2 g Coomassie Brilliant blue R- 250, 400 ml C2H5OH and 50 ml CH3COOH and make up to 1 L with deionized H2O.
    19. Washing solution
      40% C2H5OH
      5% CH3COOH
    20. HRP Color Development Solution
      Dissolve 60 mg of 4-chloro-naphtol into 20 ml of methanol.
      Disolve immediately before use and protect solution from light.
      Immediately prior to use, add 60 μl of ice cold 30% H2O2 to 100 ml TBS. Mix both solutions at RT. Use immediately.

    References

    1. Chankova, S., Mitrovska, Z., Miteva, D., Oleskina, Y. P. and Yurina, N. P. (2013a). Heat shock protein HSP70B as a marker for genotype resistance to environmental stress in Chlorella species from contrasting habitats. Gene 516(1): 184-189.
    2. Chankova, S., Mitrovska, Z. and Yurina, N. (2013b). Heat Shock Treatment of Chlamydomonas reinhardtii and Chlorella Cells. Bio-protocol 3(15): e849.
    3. Chankova, S. G., Yurina, N. P., Dimova, E. G., Ermohina, O. V., Oleskina, Y. P., Dimitrova, M. T. and Bryant, P. E. (2009). Pretreatment with heat does not affect double-strand breaks DNA rejoining in Chlamydomonas reinhardtii. J Thermal Biol 34(7): 332-336.

简介

蛋白质印迹允许目标抗体对蛋白质的特异性检测。 该方案在凝胶电泳之前利用用于小球藻的总蛋白质方案的分离。 电泳后,所选择的抗体用于检测和定量叶绿体HSP70B的水平。

材料和试剂

  1. 物种 
    使用三种小球藻物种:<普通,从利文斯顿岛,南设得兰群岛,南极的土壤样品中分离; C。 vulgaris 菌株8/1,于1968年从保加利亚Rupite地区的温泉泉中分离,并自1975年在我们的实验室中栽培,并且从Trebon收集中孵化出嗜热菌。
    栽培 
    在60μmol/m 2 s/s的连续光下和在23℃±0.1℃的温度下,在TAP(Tris Acetate Phosphate)培养基中培养小球藻属物种Phytotron GC 400生长室。该种在该温度下培养,因为众所周知,热的藻类可以在宽的温度范围内生长。
  2. 兔多克隆抗体HSP70B细胞质(Agrisera,目录号:AS06 175)
  3. 山羊抗兔IgG(H& I)HRP缀合的(Agrisera,目录号:AS09 602)
  4. 考马斯亮蓝G 250
  5. 正磷酸(Valerus,目录号:N 4420)
  6. 三氯乙酸(TCA)
  7. 牛血清白蛋白(BSA)(Applichem GmbH,目录号:1391 0025)
  8. 白蛋白级分V(pH 7.0)
  9. 中纯硝酸纤维素(NCM)(0.45μm)(Bio-Rad Laboratories,目录号:162-0115)
  10. 过滤纸
  11. 海绵
  12. 4CN(4-氯 - 萘酚)(Bio-Rad Laboratories,目录号:N170-6535)
  13. N,N',N'-四甲基乙二胺(TEMED)(Alfa Aesar,目录号:N12536)
  14. Laemmli样品缓冲液(参见配方)
  15. 布拉德福的试剂(见食谱)
  16. 5x Laemmli缓冲区(参见配方)
  17. 运行缓冲区(参见配方)
  18. 传输缓冲区(请参阅配方)
  19. SDS-PAGE凝胶(参见配方)
  20. 30%丙烯酰胺/N,N' - 亚甲基双丙烯酰胺(AA/MBA)(参见配方)< br /
  21. 10%SDS(见配方)
  22. 10%过硫酸铵(见配方)
  23. 1.5 M Tris HCl缓冲液(pH 8.8)(参见配方)
  24. 1.0 M Tris HCl缓冲液(pH 6.8)(参见配方)
  25. 4 M NaCl(见配方)
  26. 1.0 M Tris HCl缓冲液(pH 7.5)(参见配方)
  27. 20%Tween 20(见配方)
  28. 阻止缓冲区(参见配方)
  29. 染色溶液(见配方)
  30. 5%CH 3 COOH(参见配方)
  31. 洗涤液(见配方)
  32. 50 mM TBS-T缓冲液(见配方)
  33. HRP颜色开发解决方案(参见配方)

设备

  1. 电机
  2. 硅胶 石英砂0.6mm(Valerus,目录号:N1760)
  3. 离心机(Sigma-Aldrich,型号:1-15K)
  4. 电泳室转移单元Hoefer miniVE电泳和电转移单元(Hoefer,型号:SE300-10A-1.0)
  5. 迷你摇床振动器MR-1

软件

  1. Image J程序

程序


I.   细胞裂解

  1. 向沉淀物中加入100μl的Lisys溶液(LS)(Chankova等人,2013b)转移至冷冻的研钵,加入两个硅砂沙,在研钵中研磨3分钟,加入200μl LS在砂浆中洗涤并将材料转移到2ml的Eppendorf管中。
  2. 将步骤1的材料以14,500×g离心10分钟。
  3. 分离上清液,在t = 90°C加热上清液5分钟。
  4. 在14,500×g离心5分钟。
  5. 将上清液分成2个样品:第一个用于测定蛋白质浓度; 第二个保持在t = -20°С。


II。  蛋白质浓度的测定(Bradford)

  1. 加入30μl20%TCA到30μl上清液。
  2. 在14,500×g离心5分钟。
  3. 向沉淀中加入60μl0.1N NaOH,充分混合。 为了获得最佳结果,加入30μl0.1N NaOH两次。
  4. 取14μl,加入86μl0.15μlNaCl和3 ml Bradford试剂。
  5. 使用蛋白质量的校准曲线(表1) 对于校准曲线:
    储备溶液 - 0.5mg/ml BSA
    使用表1确定每个点的标准曲线加入3ml试剂的Bradford。

    表1.蛋白质量的校准曲线
    N
    BSA(μg)
    BSA(0.5mg/ml)vol(μl)
    NaCl(0.15M)vol(μl)
    1
    0
    0
    100
    2
    5
    10
    90
    3
    10
    20
    80
    4
    15
    30
    70
    5
    20
    40
    60
    6
    25
    50
    50
    7
    30
    60
    40
    8
    35
    70
    30
    9
    40
    80
    20
    10
    45
    90
    10
    11
    50
    100
    0

III。 蛋白电泳

  1. 将大约100 ml的1x Laemmli缓冲液放入电泳模块的比色杯中。
  2. 取出梳子,用SDS-PAGE凝胶缓冲液冲洗孔。
  3. 每孔加入10μg蛋白质:调节体积,加入等量的蛋白质(例如:10μl样品中含有10μg蛋白质)。
  4. 将剩余的缓冲液放入电泳槽的浴槽中(体积必须始终高于最小值)。
  5. 使用以下参数进行电泳:120V和16mA,3.5小时
  6. 电泳完成后,小心取出凝胶。
    注意:删除样本的顺序。 应释放浓缩凝胶。


IV。 蛋白质在NCM上的转移

  1. 在缓冲液中浸泡凝胶15分钟。
  2. 在转移缓冲液中浸泡海绵和三明治的滤纸
  3. 剪切NCM。 尺寸应该是诸如凝胶的尺寸。 将NCM在转移缓冲液中放置5分钟。
    注意:标记膜上样品的顺序! 用铅笔标记膜。
  4. 做一个三明治。
    1. 该堆叠组装在黑色阴极侧(参见图1):
      1. 将包装海绵置于黑色阴极侧。
      2. 将包装海绵放置在黑色catode(a)上。
      3. 在海绵上放置一张湿滤纸(b)。
      4. 将平衡凝胶放在滤纸(c)上。
      5. 将膜置于凝胶(d)上。
      6. 在膜(e)上放置一张湿滤纸。
      7. 在滤纸(f)上放置两个包装海绵。
      8. 如果添加了第二个转移堆叠,则放置在这两个海绵之间。


        图1. 组装转移堆栈( t his是从www。hoeferinc.com )

    2. 不同部位的三明治压力很好,以避免气泡。
    3. 三明治的不同部分应该很好地润湿。 你可以用管子"滚动"他们。
      1. 关闭设备。 放入腔室转移缓冲液。
      2. 使用以下参数进行印迹:35V和250mA,持续2小时


V.  蛋白质印迹

  1. 蛋白转移后,将膜置于封闭缓冲液中t = 4°C。 在摇摆平台上孵育1小时(按照此步骤,我们获得了最佳结果)。
  2. 将凝胶放在染色溶液中4-5小时
  3. 用洗涤液洗涤凝胶3-4小时。
  4. 在封闭缓冲液(1:10,000)稀释初级抗体,根据制造商的说明孵育。 在摇床平台上在t = 4℃过夜孵育
  5. 以下列方式在摇床平台上的TBS-T缓冲液中洗膜:2分钟(2×2分钟),2次,10分钟(2×10分钟)。
  6. 在封闭缓冲液(1:20,000)中制备二抗,并根据制造商的说明进行孵育。 在室温下摇动平台孵育2小时。
  7. 在摇床平台上的TBS-T缓冲液中以以下方式洗涤膜:两次2分钟(2×2分钟),然后三次,5分钟(3×5分钟)。
  8. 使用HRP显色解决方案,4CN根据制造商的说明可视化。
  9. 扫描膜。 使用Image J程序计算蛋白质量。

食谱

  1. Laemmli样品缓冲区
    2%SDS
    5%2-巯基乙醇 10%甘油 0.002%(w/v)溴酚蓝
    62.5mM Tris HCl(pH6.8)
  2. Bradford的试剂
    在搅拌器中溶解100mg考马斯亮蓝G 250和50ml 96%的醇15分钟。 加入94.5ml 90%正磷酸。
    加入900ml去离子H 2 O并轻轻搅拌。
    通过折叠的滤纸过滤,并用去离子水补足至1L。
    保存在冰箱中t = 4°C。
  3. 5x Laemmli缓冲区
    15克TRIS基地
    72g甘氨酸在1L去离子H 2 O中
  4. 运行缓冲区
    将200ml 5x Laemmli缓冲液+ 10ml 10%SDS加入到1L去离子H 2 O中。
  5. 传输缓冲区
    将200ml 5x Laemmli缓冲液+ 2ml 10%SDS加至+ 200ml乙醇至1L去离子H 2 O中。
  6. SDS-PAGE凝胶
    注意:在制备SDS-PAGE凝胶之前,玻璃砖应用酒精彻底清除。
    1. 分离凝胶(12.5%)(表2)

      表2.制备分离凝胶溶液
      微型凝胶数量
      1
      2
      去离子H 2 O 2 / 3.2 ml
      6.4 ml
      Acrilamide/bisacrilamide(30%)
      4 ml
      8.0 ml
      1.5M Tris HCl缓冲液,pH 8.8
      2.6 ml
      5.2 ml
      10%SDS
      100微升
      200μl
      10%APS
      100微升
      200μl
      TEMED
      10微升
      20微升

      1. 非常仔细地混合组分在一个50ml Falcon管,以避免气泡。
      2. 在室的两块玻璃板之间插入分离凝胶(在砖的边界下约1cm)
      3. 使用注射器小心地加入去离子H 2 O 2作为薄膜,并等待约15分钟
      4. 小心取出水; 用滤纸擦拭两端的水滴。
    2. 堆叠凝胶(表3)

      表3.准备4%堆积胶溶液
      微型凝胶数量
      1
      2
      去离子H 2 O 2 / 1,370μl
      2,740μl
      Acrilamide/bisacrilamide(30%)
      330微升
      660μl
      1.0M Tris HCl缓冲液,pH 6.8
      250微升
      500微升
      10%SDS
      20微升
      40微升
      10%APS
      20微升
      40微升
      TEMED
      2微升
      4微升

    3. 放入浓缩的凝胶,插入梳子,等待凝胶聚合。
    4. 对于电泳更好是准备约1,250ml 1x Laemmli缓冲液。 它可以使用两次。
  7. 30%AA/MBA
    将29.0g + 1.0g MBA溶解在72.5ml去离子H 2 O中,使体积达到100ml,使用0.45μm过滤器进行过滤
    保持在t = 4°C小于1个月。
  8. 10%SDS
    将10g SDS溶解在100ml去离子H 2 O中
  9. 10%过硫酸铵
    将1g溶解在10ml去离子H 2 O中 保持在t = 4°C小于1个月。
  10. 1.5M Tris HCl缓冲液,pH 8.8
    将18.5g Tris碱溶解在80ml去离子H 2 O中,用浓HCl调节至pH = 8.8,并将体积补足至100ml。
  11. 1.0 M Tris HCl缓冲液pH 6.8
    将12.114g Tris碱溶解在80ml去离子H 2 O中,用浓HCl调节至pH = 6.8,并将体积补至100ml。
  12. 50mM TBS-T缓冲液
    1.0M Tris HCl缓冲液(pH7.5) 200 mM NaCl
    0.1%Tween 20
  13. 4 M NaCl
    将23.376g NaCl溶解在100ml去离子H 2 O中
  14. 1.0M Tris HCl缓冲液(pH7.5) 将12.114g TRIS碱溶解在80ml去离子H 2 O中,用浓HCl调节至pH7.5,并补足至100ml。
  15. 20%Tween 20
    将20ml吐温补足至100ml去离子H 2 O
  16. 阻塞缓冲区
    将5%无脂奶粉溶于100 ml TBS-T缓冲液中。
  17. 染色溶液
    0.2%考马斯亮蓝R-250
    40%C 2 H 5 OH
  18. 5%CH 3 COOH v/v 将2g考马斯亮蓝R-250,400ml C 2 H 5 OH和50ml CH 3 COOH溶解并补足至1L 用去离子H 2 O 2
  19. 洗涤溶液
    40%C 2 H 5 OH
    5%CH 3 COOH v/v
  20. HRP色彩开发解决方案
    将60mg的4-氯 - 萘酚溶于20ml甲醇中。
    使用前立即溶解并保护溶液免受光照。
    在即将使用前,向60ml TBS中加入60μl冰冷的30%H 2 O 2 Sub 2。 在室温下混合两种溶液。 立即使用。

参考文献

  1. Chankova,S.,Mitrovska,Z.,Miteva,D.,Oleskina,Y.P.和Yurina,N.P。(2013a)。 热休克蛋白HSP70B作为基因型对环境压力的标记,在小球藻中 >来自对比生境的物种。 Gene 516(1):184-189。
  2. Chankova,S.,Mitrovska,Z.和Yurina,N。(2013b)。热休克治疗 (Chlamydomonas reinhardtii)和小球藻细胞。生物协议 3(15):e849。
  3. Chankova,S.G.,Yurina,N.P.,Dimova,E.G.,Ermohina,O.V.,Oleskina,Y.P.,Dimitrova,M.T.and Bryant,P.E。(2009)。 用热预处理不会影响双链断裂重新结合在莱茵衣藻中的DNA 。 J Thermal Biol 34(7):332-336。
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引用:Chankova, S., Mitrovska, Z. and Yurina, N. (2013). Western Blot Analysis of Chloroplast HSP70B in Chlorella Species . Bio-protocol 3(15): e850. DOI: 10.21769/BioProtoc.850.
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