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2D Diagonal Redox SDS-PAGE of Proteins
蛋白质二维双向还原电泳技术   

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Abstract

2D diagonal redox SDS-PAGE of proteins is used to detect intramolecular or intermolecular disulfide bridges using Chlamydomonas in this example (Stroeher and Dietz, 2008; Schwarz et al., 2012). Both dimensions consist of a conventional SDS-PAGE, except that the sample buffer for the first dimension lacks a reducing agent. Intermolecular disulfide bridges increase the apparent molecular weight of a protein in the first dimension, whereas intramolecular bridges decrease the apparent weight of the protein.

Keywords: 2d Diagonal SDS PAGE(二维对角线电泳), Redox(氧化还原), Disulfide bridge(二硫桥)

Materials and Reagents

  1. All material for a conventional Laemmli SDS-PAGE
  2. Non-reducing Laemmli sample buffer
  3. KCl (Applichem, catalog number: A1039 )
  4. Triton X-100 (Applichem, catalog number: A1388 )
  5. DTT (Applichem, catalog number: A1101 )
  6. SDS (SERVA Electrophoresis GmbH, catalog number: 20765 )
  7. Acrylamide 40% 37.5:1 (SERVA Electrophoresis GmbH, catalog number: 10681 )
  8. Ammoniumperoxodisulfate (Carl Roth, catalog number: 9592 )
  9. TEMED (Carl Roth, catalog number: 2367 )
  10. Beta-mercaptoethanol (Carl Roth, catalog number: 4227 )
  11. Iodoacetamide (Applichem, catalog number: A1666 )
  12. Tris (Applichem, catalog number: A1379 )
  13. Na2EDTA (Carl Roth, catalog number: 8043 )
  14. Tricine (Carl Roth, catalog number: 6977 )
  15. Glycine (SERVA Electrophoresis GmbH, catalog number: 23390 )
  16. Agarose (SERVA Electrophoresis GmbH, catalog number: 11380 )
  17. Bromophenol blue (Carl Roth, catalog number: T116 )
  18. Filter paper (Munktell, catalog number: 2.519.580600N )
  19. Page Ruler unstained protein ladder (Thermo Fisher Scientific, catalog number: 26614 )
  20. 2x Cell lysis buffer (see Recipes)
  21. 5x non-reducing Laemmli sample buffer (see Recipes)
  22. 5x reducing Laemmli sample buffer (see Recipes)
  23. Laemmli electrophoresis buffer (see Recipes)

Equipment

  1. All equipment for a conventional Laemmli SDS-PAGE

Procedure

  1. Cell lysis (perform all lysis steps at 4 °C)
    1. Break your cells with 2x cell lysis buffer (use 1 ml of lysis buffer per pellet of a 1 L culture) by repeated pipetting for up to 5 min.
    2. Solubilize proteins with a final concentration of 1% SDS (30 min, 4 °C, no agitation required).
    3. Prevent thiol-reshuffling of your sample by alkylation with a final concentration of 0.1 M iodoacetamide (30 min, 4 °C, in the dark as iodoacetamide is not stable in light, no agitation required).
  2. First dimension (keep in mind that each lane of the first dimension requires a separate gel for the second dimension when calculating the number of samples that you want to process)
    1. Add appropriate amount of non-reducing Laemmli sample buffer to each sample while staying within the size limits of your gel comb, use enough material to enable the analysis of your protein by your detection method.
    2. Denature sample according to the best conditions for your protein (e.g. many soluble proteins can be denatured at 95 °C for 6 min).
    3. Perform Laemmli SDS-PAGE with your sample (Figure 1).
  3. Second dimension
    1. Excise lanes from gel as gel stripes (Figure 1).
    2. Reduce disulfide bridges by agitating your gel stripes in an amount of Laemmli electrophoresis buffer that covers your gel stripe completely (+ 0.1 M DTT [final concentration], 15 min, room temperature).
    3. Prevent thiol-reshuffling through alkylation by agitating your gel stripes in Laemmli electrophoresis buffer (containing 0.1 M idoacetamide [final concentration], 15 min, room temperature, in the dark).
    4. Prepare a separate gel for the 2nd dimension SDS-PAGE of each gel stripe with a shorter stacking gel (approximately 50% shorter than your stacking gel in the first dimension and no wells) to accommodate the gel stripe of the first dimension into your gel apparatus.
    5. Place gel stripe from first dimension in horizontal orientation (higher molecular weight on the left side, lower molecular weight on the right side) above stacking gel of the 2nd dimension (Figure 1).
    6. Boil 0.5% agarose (in Laemmli electrophoresis buffer with bromophenol blue [0.75 g L-1]) and fill your gel of the second dimension with the agarose solution (Figure 1).
    7. Press gel stripe against stacking gel of the second dimension (do not change orientation of gel stripe, avoid or remove air bubbles between gel stripe and stacking gel) before agarose solidifies (Figure 1).
    8. Pipet protein size marker on a small stripe of filter paper and stick that piece of paper beside your gel stripe into the agarose solution before agarose solidifies.
    9. Run Laemmli SDS-PAGE as the second dimension (bromophenol blue from the solidified agarose serves as running dye, a 17 cm gel of 1.5 mm thickness requires ~16 h at 14 mA) (Figure 1).
    10. Analyze your protein of interest (e.g. western blotting, MS).
    11. Proteins with intermolecular disulfide bridges appear on the left side of the diagonal, as their apparent weight shrank from the first to the second dimension.
    12. Proteins without disulfide bridges appear directly on the diagonal of your 2D PAGE, as there is no difference in their electrophoresis pattern between the two dimensions.
    13. Proteins with intramolecular disulfide bridges appear on the right side of the diagonal, as their apparent weight increased from the first to the second dimension.


      Figure 1. A scheme of the steps 2c - 3i with the results for the proteins described in 3k - 3m.

Recipes

  1. 2x cell lysis buffer
    ~200 ml dH2O
    2.24 g KCl
    97 mg EDTA
    0.89 g Tricine
    2.5 ml Triton X-100
    Adjust pH to 7.8
    Fill up with dH2O to 250 ml
  2. 5x non-reducing Laemmli sample buffer
    ~40 ml dH2O
    1.51 g Tris
    3.75 g SDS
    0.125 g Bromophenol blue
    Adjust pH to 6.8
    Fill up with dH2O to 50 ml
  3. 5x reducing Laemmli sample buffer
    Add 0.44 ml beta-mercaptoethanol to 50 ml of 5x non-reducing Laemmli sample buffer
  4. Laemmli electrophoresis buffer
    ~800 ml dH2O
    3.03 g Tris
    14.41 g glycine
    1.5 g SDS
    Fill up with dH2O to 1,000 ml

References

  1. Schwarz, C., Bohne, A. V., Wang, F., Cejudo, F. J. and Nickelsen, J. (2012). An intermolecular disulfide-based light switch for chloroplast psbD gene expression in Chlamydomonas reinhardtii. Plant J 72(3): 378-389.
  2. Stroher, E. and Dietz, K. J. (2008). The dynamic thiol-disulphide redox proteome of the Arabidopsis thaliana chloroplast as revealed by differential electrophoretic mobility. Physiol Plant 133(3): 566-583. 

简介

在该实施例中,使用蛋白质的2D对角线氧化还原SDS-PAGE用于检测分子内或分子间二硫键(Stroeher和Dietz,2008; Schwarz等人,2012) 。 两个维度由常规SDS-PAGE组成,除了第一维的样品缓冲液不含还原剂。 分子间二硫桥在第一维中增加蛋白质的表观分子量,而分子内桥降低蛋白质的表观重量。

关键字:二维对角线电泳, 氧化还原, 二硫桥

材料和试剂

  1. 常规Laemmli SDS-PAGE的所有材料
  2. 非还原Laemmli样品缓冲液
  3. KCl(Applichem,目录号:A1039)
  4. Triton X-100(Applichem,目录号:A1388)
  5. DTT(Applichem,目录号:A1101)
  6. SDS(SERVA Electrophoresis GmbH,目录号:20765)
  7. 丙烯酰胺40%37.5:1(SERVA Electrophoresis GmbH,目录号:10681)
  8. 过氧二硫酸铵(Carl Roth,目录号:9592)
  9. TEMED(Carl Roth,目录号:2367)
  10. β-巯基乙醇(Carl Roth,目录号:4227)
  11. 碘乙酰胺(Applichem,目录号:A1666)
  12. Tris(Applichem,目录号:A1379)
  13. Na 2 EDTA(Carl Roth,目录号:8043)
  14. Tricine(Carl Roth,目录号:6977)
  15. 甘氨酸(SERVA Electrophoresis GmbH,目录号:23390)
  16. 琼脂糖(SERVA Electrophoresis GmbH,目录号:11380)
  17. 溴酚蓝(Carl Roth,目录号:T116)
  18. 滤纸(Munktell,目录号:2.519.580600N)
  19. 页面尺度未染色蛋白梯度(Thermo& Fisher Scientific,目录号:26614)
  20. 2x细胞裂解缓冲液(见Recipes)
  21. 5x非还原Laemmli样品缓冲液(参见配方)
  22. 5x减少Laemmli样品缓冲液(见配方)
  23. Laemmli电泳缓冲液(参见配方)

设备

  1. 所有设备的常规Laemmli SDS-PAGE

程序

  1. 细胞裂解(在4℃下进行所有裂解步骤)
    1. 用2x细胞裂解缓冲液(使用1ml裂解缓冲液/1L培养物的沉淀)破碎细胞,重复移液至多5分钟。
    2. 溶解蛋白质,终浓度为1%SDS(30分钟,4℃,无需搅拌)
    3. 通过最终浓度为0.1M碘乙酰胺(30分钟,4℃,在黑暗中,因为碘乙酰胺在光照下不稳定,不需要搅拌)的烷基化来防止样品的巯基重排。
  2. 第一维(请记住,在计算要处理的样本数时,第一维的每个泳道都需要单独的第二维凝胶)
    1. 在每个样品中加入适量的非还原Laemmli样品缓冲液,同时保持在凝胶梳的尺寸范围内,使用足够的材料,通过检测方法分析蛋白质。
    2. 根据蛋白质的最佳条件使样品变性(例如,许多可溶性蛋白质可以在95℃变性6分钟)。
    3. 用您的样品进行Laemmli SDS-PAGE(图1)
  3. 第二维
    1. 消毒道从凝胶上作为凝胶条纹(图1)。
    2. 通过在一定量的Laemmli电泳缓冲液中搅拌您的凝胶条来减少二硫键,所述电泳缓冲液完全覆盖了凝胶条(+ 0.1M DTT [最终浓度],15分钟,室温)。
    3. 通过在Laemmli电泳缓冲液(含有0.1M偶氮乙酰胺[最终浓度],15分钟,室温,在黑暗中)搅拌凝胶条,通过烷基化防止硫醇重新洗牌。
    4. 使用较短的堆积凝胶(比第一维中的堆叠凝胶短约50%,没有孔)制备用于每个凝胶条的第二层和第二层SDS-PAGE的单独的凝胶以适应凝胶条 的第一维进入你的凝胶装置
    5. 将凝胶条从第一维在水平方向(在左侧的较高分子量,在右侧的较低分子量)放置在第二尺寸的堆积凝胶上方(图1)。
    6. 煮0.5%琼脂糖(在具有溴酚蓝的Laemmli电泳缓冲液中[0.75g L -1 -1 ]),并用琼脂糖溶液填充第二维的凝胶(图1)。
    7. 在琼脂糖凝固之前,将凝胶条压在第二维的堆积凝胶上(不要改变凝胶条的方向,避免或去除凝胶条和凝胶之间的气泡)(图1)。
    8. Pipet蛋白质大小标记在一小条滤纸上,并在琼脂糖凝固之前,将您的凝胶条纹旁边的那张纸粘在琼脂糖溶液中。
    9. 运行Laemmli SDS-PAGE作为第二维(来自固化的琼脂糖的溴酚蓝用作流动染料,15cm厚度的17cm凝胶需要在14mA下约16小时)(图1)。
    10. 分析您感兴趣的蛋白质(例如, 免疫印迹,MS)
    11. 具有分子间二硫桥的蛋白质出现在对角线的左侧,因为它们的表观重量从第一维缩小到第二维。
    12. 没有二硫键的蛋白质直接出现在2D PAGE的对角线上,因为它们在两个维度之间的电泳图案没有差别。
    13. 具有分子内二硫桥的蛋白质出现在对角线的右侧,因为它们的表观重量从第一维增加到第二维。


      图1.具有3k-3m中描述的蛋白质结果的步骤2c-3i的方案。

食谱

  1. 2x细胞裂解缓冲液
    〜200ml dH 2 O
    2.24克KCl
    97mg EDTA
    0.89g Tricine
    2.5ml Triton X-100 将pH调节至7.8
    用dH <2> O填充至250ml
  2. 5x非还原Laemmli样品缓冲液
    〜40ml dH 2 O
    1.51克Tris
    3.75克SDS
    0.125g溴酚蓝
    将pH调节至6.8
    加满dH 2 2至50ml
  3. 5x减少Laemmli样本缓冲区
    向50ml 5x非还原Laemmli样品缓冲液
    中加入0.44mlβ-巯基乙醇
  4. Laemmli电泳缓冲液
    〜800ml dH 2 O
    3.03g Tris
    14.41g甘氨酸
    1.5克SDS
    填充dH 2 2至1000ml

参考文献

  1. Schwarz,C.,Bohne,A.V.,Wang,F.,Cejudo,F.J.and Nickelsen,J。(2012)。 在莱茵衣藻中的叶绿体psbD基因表达的分子间二硫键光开关。 Plant J 72(3):378-389。
  2. Stroher,E。和Dietz,K.J。(2008)。 显示拟南芥叶绿体的动态硫醇 - 二硫化物氧化还原蛋白质组 通过差别电泳迁移率。 生理植物 133(3):566-583。 
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Schwarz, C. and Nickelsen, J. (2013). 2D Diagonal Redox SDS-PAGE of Proteins. Bio-protocol 3(11): e781. DOI: 10.21769/BioProtoc.781.
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