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Semi-denaturing Detergent Agarose Gel Electrophoresis (SDD-AGE)
半变性去垢剂琼脂糖凝胶电泳法(SDD-AGE)   

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Abstract

Pathological proteins in neurodegenerative diseases suffer a conformational change to a misfolded amyloid state. Such pathological event leads to the aggregation of these proteins that indefinitely propagates as an altered form of itself, and harbor prion-like properties (Wickner, 1994; Prusiner, 2012). In addition to diseases, prions can also have beneficial adaptive roles in lower eukaryotes (in fungi and yeast) (Eaglestone et al., 1999; True et al., 2004; Coustou et al., 1999). Besides separating polymers from their precursor soluble monomers, another particular difficulty of the study of amyloid proteins is to resolve the heterogeneity of the aggregates, since these usually exhibit a variable degree of polymorphism. Semi-denaturating detergent agarose gel electrophoresis (SDD-AGE) is a technique that takes advantage of both the property of prions and prion-like polymers to be highly resistant to solubilization by SDS detergent, and the large pores sizes of agarose, that allow the resolution of high molecular weight complexes. In this method, we describe in detail how this technique can be used to characterize heterogeneous aggregation in bacteria and yeast (Gasset-Rosa et al., 2014; Molina-García and Giraldo, 2014), and further be applied to study the aggregation pattern of proteins that become prone to aggregation through genetic manipulation.

Materials and Reagents

  1. Bacteria: Gram negative Escherichia coli (E. coli) or yeast Saccharomyces cerevisiae (S. cerevisiae)
    Notes:
    Amyloids exist in E. coli and S. cerevisiae. In both organism amyloids can be functional. In E. coli the most well know example is the curli protein which is assembled in the extracellular medium and allow the attechemnet of the bacteria to the surface and sustain the formation of biofilms (Chiti and Dobson, 2006; Chapman et al., 2002).
    In S. cerevisiae, amyloids confer to cells selctive advantage under certain physiological conditions and they aren’t harmful (Chien et al., 2004; Shorter and Lindquist, 2005; Wickner et al., 2007), a few examples are Sup35p y Ure2p, Rnq1p. The conversion of Sup35 from soluble to the amyloid state drives the reduction of translation termination activity (Wickner et al., 1995).
  2. Protease inhibitor cocktail (Roche Diagnostics, catalog number: 04 693 159 001 )
  3. Silica beads (1 mm lysing matrix C) (MP Biomedicals, catalog number: 6912-050 )
  4. Pure agarose D2 (Pronadisa, catalog number: 8034 )
  5. Detergents: SDS (Bio-Rad Laboratories, catalog number: 161-0301 ) and Sarkosyl 97% N-lauroylsarcosine (Sigma-Aldrich, catalog number: L-9150 )
  6. PVDF membrane (Bio-Rad Laboratories)
  7. Antibodies
    Notes:
    Depends on the protein you work, the antibody may be against the protein that you are studying for aggregation. A recommended strategy if the user is going to generate a recombinant protein is to include a tag in the amino or carboxyl terminal of the protein and use a specific antibody against the tag. One example is the Histidine-tag, the corresponding antibody would be a Monoclonal anti-polyhistidine (Sigma-Aldrich, catalog number: H1029 ).
    Antibodies that recognize monomer state may still be able to identify the protein in polymers even is the protein conformation has changed. Usually monomer antibodies have a broad range recognition epitopes. In our hands the antibodies used were:
    For E. coli, house-made polyclonal antibodies against RepA (generated in rabbit immunized with soluble RepA), (Gasset-Rosa et al., 2014); and monoclonal anti poly-histidine against His-RepA (WH1) (Sigma-Aldrich, catalog number: H1029) (Molina-García and Giraldo, 2014). Both of them work.
    For S. cerevisiae, anti-HA antibody (Roche Diagnostics, catalog number: 12CA5 ) against the HA (Human influenza hemagglutinin) tag of Sup35 protein (unpublished).
  8. Cell culture medium
    1. Luria Bertani (LB) medium (see Recipes)
    2. Yeast extract peptone dextrose (YPD) (see Recipes)
  9. Lysis buffer (see Recipes)
  10. Running buffer (see Recipes)
  11. Loading buffer (see Recipes)
  12. Transference buffer (see Recipes)

Equipment

  1. Fast Prep-MilliPore/MP FastPrep-24 homogenizer (catalog number: 6004-500 )
  2. Horizontal agarose electrophoresis system (Electrophoresis power supply EPS601 General Electrics)
  3. Wet/Tank transfer blotting system (Bio-Rad Laboratories)

Procedure

  1. Growth cells in your adjusted conditions.
  2. Harvest cells.
    1. Bacteria: Harvest cells (25 ml O.D. = 2) by centrifugation (10 min, 3,500 rpm, 4 °C). And, resuspend the cells in 400 μl of lysis buffer.
    2. Yeast: Harvest cells (200 ml O.D. = 0.8) by centrifugation (5 min, 3,500 rpm, 4 °C). And, resuspend the cells in 500 μl of lysis buffer.
  3. Transfer the suspension to a tube containing silica beads (usually 1/3 of the volume is sufficient).
  4. Lysate the cells.
    1. Bacteria: Lysate the cells using the Fast-Prep 24 homogenizer (4 cycles at speed IV, 30 sec each at 4 °C). And, dilute 3 μl of cell lysate with 30 μl with lysis buffer, and then add 10 μl of 4x loading buffer to the sample.
    2. Yeast: Lysate the cells using the Fast-Prep 24 homogenizer (5 cycles at speed V, 30 sec each at 4 °C). And, add 10 μl of 4x loading buffer to 30 μl of the lysate.
  5. Incubate the samples 10 min at room temperature.
  6. Load them in the agarose gel.
    1. Prepare 200 ml of agarose at 1.5% in 1x TAE.
      Note: Heat the agarose suspension to melting and then add SDS to 0.1%.
    2. Pour it into 20 cm x 24 cm horizontal plate mould. Make sure that the casting tray is on a flat surface.
      Notes:
      1. Eliminate all the possible bubbles.
      2. Agarose electrophoresis are run using horizontal agarose electrophoresis system, the samples need to be run for long distance in order to resolve polymers of high molecular weight.
    3. Fit a thick comb (e.g., >2 mm, 20-well) allowing to load 40 μl per sample.
    4. Submerge the gel in a horizontal electrophoresis container with 1x TAE-0.1% SDS at 10 °C.
  7. Run electrophoresis at 100 V for 7.5 h at 10 °C.
    Note: For detecting oligomers with high hydrophobic feature run it at lower voltage 50 V and room temperature for 12 h with 1% (final concentration) of Sarkosyl in the sample (4% Sarkosyl in the loading buffer). The concentration for the running buffer is TAE 1x and 0.1% SDS as described before.
    Caution: Extreme care during electrophoresis to avoid any electrical hazard.
  8. Cut the PVDF membrane adjusted to the size of the agarose gel and 2 pieces with the same dimensions of Whatman paper.
  9. Attach tightly the gel to the PDVF membrane and add in each side a piece of Whatman paper.
    Note: Everything should be pre-wet with transference buffer. Make sure that there are no bubbles in between.
  10. Place the cast into the Wet Transfer-Blot and cover with buffer 1x TAE-0.1% SDS.
    Caution: Extreme care during electrophoresis to avoid any electrical hazard.
  11. Run the transference at 16 V/400 mA and 10 °C for 15 h.
  12. Perform Western-blot with the appropriate antibodies to detect your polymers.

Recipes

  1. Luria Bertani (LB) medium
    Bacto-tryptone 10 g/L
    Yeast extract 5 g/L
    NaCl 5 g/L
  2. Yeast extract peptone dextrose (YPD)
    2% bacto peptone
    2% glucose
    1% yeast extract
  3. Lysis buffer
    25 mM Tris-HCl (pH 6.8)
    250 mM NaCl
    5 mM EDTA
    10% glycerol supplemented with protease inhibitors (1 tablet each 10 ml)
  4. Running buffer
    1x TAE (Tris acetate EDTA)
    0.1% SDS
  5. Loading buffer (4x)
    0.5% TAE
    5% glycerol
    2% sarkosyl
    0.1 mg/ml bromophenol blue plus protease inhibitors (1 tablet each 10 ml)
  6. Transference buffer
    1x TAE (Tris Acetate EDTA)
    0.1% SDS

Acknowledgments

We thank Prof. Rafael Giraldo for his encouragement and continuous support, and Spanish MINECO for funding (BIO2012-30852 and CSD2009-00088).

References

  1. Coustou, V., Deleu, C., Saupe, S. J. and Begueret, J. (1999). Mutational analysis of the [Het-s] prion analog of Podospora anserina. A short N-terminal peptide allows prion propagation. Genetics 153(4): 1629-1640.
  2. Eaglestone, S. S., Cox, B. S. and Tuite, M. F. (1999). Translation termination efficiency can be regulated in Saccharomyces cerevisiae by environmental stress through a prion‐mediated mechanism. EMBO J 18(7): 1974-1981.
  3. Gasset-Rosa, F., Coquel, A. S., Moreno-Del Alamo, M., Chen, P., Song, X., Serrano, A. M., Fernandez-Tresguerres, M. E., Moreno-Diaz de la Espina, S., Lindner, A. B. and Giraldo, R. (2014). Direct assessment in bacteria of prionoid propagation and phenotype selection by Hsp70 chaperone. Mol Microbiol 91(6): 1070-1087.
  4. Molina-García, L. and Giraldo, R. (2014). Aggregation interplay between variants of the RepA-WH1 prionoid in Escherichia coli. J Bacteriol 01527-01514.
  5. Prusiner, S. B. (2012). Cell biology. A unifying role for prions in neurodegenerative diseases. Science 336(6088): 1511-1513.
  6. True, H. L., Berlin, I. and Lindquist, S. L. (2004). Epigenetic regulation of translation reveals hidden genetic variation to produce complex traits. Nature 431(7005): 184-187.
  7. Wickner, R. B. (1994). [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science 264(5158): 566-569.

简介

神经退行性疾病中的病理蛋白质遭受错误折叠的淀粉样蛋白状态的构象变化。这种病理性事件导致这些蛋白质的聚集,其作为其自身的改变形式无限地繁殖,并且具有朊病毒样特性(Wickner,1994; Prusiner,2012)。除了疾病,朊病毒还可以在低等真核生物(真菌和酵母)中具有有益的适应性作用(Eaglestone等人,1999; True等人,2004; Coustou等人,1999)。除了从其前体可溶性单体中分离聚合物之外,研究淀粉样蛋白的另一个特别困难是解决聚集体的异质性,因为这些通常表现出不同程度的多态性。半变性洗涤剂琼脂糖凝胶电泳(SDD-AGE)是一种利用朊病毒和朊病毒样聚合物的性质来高度抵抗SDS洗涤剂的溶解的技术,以及琼脂糖的大孔径,其允许分辨率的高分子量复合物。在该方法中,我们详细描述了该技术如何用于表征细菌和酵母中的异质聚集(Gasset-Rosa等人,2014; Molina-García和Giraldo,2014)应用于研究通过遗传操作变得易于聚集的蛋白质的聚集模式。

材料和试剂

  1. 细菌:革兰氏阴性大肠杆菌(大肠杆菌)或酵母酿酒酵母(酿酒酵母 /> 注意:
    淀粉样蛋白存在于大肠杆菌和酿酒酵母中。在两种生物体中,淀粉样蛋白可以是功能性的。在大肠杆菌中,最知名的实例是卷曲蛋白,其在胞外培养基中组装并允许细菌的表面细胞到表面并维持生物膜的形成(Chiti和Dobson,2006; Chapman等人,2002) 。
    在酿酒酵母中,淀粉样蛋白在某些生理条件下赋予细胞选择优势并且它们不是有害的(Chien等人,2004; Shorter和Lindquist,2005; Wickner等人,2007),一些实例是Sup35p y Ure2p,Rnq1p。 Sup35从可溶性转变为淀粉样蛋白状态推动了翻译终止活性的降低(Wickner等,1995)。
  2. 蛋白酶抑制剂混合物(Roche Diagnostics,目录号:04 693 159 001)
  3. 硅胶珠(1mm裂解基质C)(MP Biomedicals,目录号:6912-050)
  4. 纯琼脂糖D2(Pronadisa,目录号:8034)
  5. 洗涤剂:SDS(Bio-Rad Laboratories,目录号:161-0301)和Sarkosyl 97%N-月桂酰肌氨酸(Sigma-Aldrich,目录号:L-9150)
  6. PVDF膜(Bio-Rad Laboratories)
  7. 抗体
    注意:
    取决于您工作的蛋白质,抗体可能针对您正在研究聚集的蛋白质。如果使用者要产生重组蛋白,推荐的策略是在蛋白的氨基或羧基末端包括标签,并使用针对标签的特异性抗体。一个实例是组氨酸标签,相应的抗体是单克隆抗多组氨酸(Sigma-Aldrich,目录号:H1029)。
    识别单体状态的抗体仍然能够鉴定聚合物中的蛋白质,即使蛋白质构象已经改变。通常单体抗体具有广泛的识别表位。在我们手中,使用的抗体是:
    对于大肠杆菌,制备了针对RepA(在用可溶性RepA免疫的兔中产生的)的自制多克隆抗体,(Gasset-Rosa等人,2014);和抗His-RepA(WH1)(Sigma-Aldrich,目录号:H1029)的单克隆抗多组氨酸(Molina-García和Giraldo,2014)。两者都有效。
    对于酿酒酵母,针对Sup35蛋白(未公开)的HA(人流感血凝素)标记的抗HA抗体(Roche Diagnostics,目录号:12CA5)。
  8. 细胞培养基
    1. Luria Bertani(LB)培养基(见配方)
    2. 酵母提取物蛋白胨葡萄糖(YPD)(见Recipes)
  9. 裂解缓冲液(见配方)
  10. 运行缓冲区(参见配方)
  11. 加载缓冲区(参见配方)
  12. 传输缓冲区(请参阅配方)

设备

  1. Fast Prep-MilliPore/MP FastPrep-24匀浆器(目录号:6004-500)
  2. 水平琼脂糖电泳系统(电泳电源EPS601通用电气公司)
  3. 湿/罐转移印迹系统(Bio-Rad Laboratories)

程序

  1. 在调整条件下的生长细胞
  2. 收获细胞。
    1. 细菌:通过离心收获细胞(25ml O.D. = 2)(10分钟, 3500rpm,4℃)。 然后,将细胞重悬在400μl裂解缓冲液中
    2. 酵母:通过离心(5分钟,10分钟)收获细胞(200ml O.D. = 0.8) 3500rpm,4℃)。 并且,将细胞重悬在500μl裂解缓冲液中。
  3. 将悬浮液转移到含有二氧化硅珠的管中(通常1/3体积就足够了)。
  4. 裂解细胞。
    1. 细菌:使用Fast-Prep 24匀浆器裂解细胞(4个循环 在4℃下,每次30秒)。 并且,用3μl的细胞裂解物稀释 30μl用裂解缓冲液,然后加入10μl4x上样缓冲液 样品
    2. 酵母:使用Fast-Prep 24匀浆器裂解细胞(在速度V下5个循环,每个在4℃下30秒)。 并且,向30μl裂解物中加入10μl的4x上样缓冲液。
  5. 在室温下孵育样品10分钟。
  6. 将它们装入琼脂糖凝胶中
    1. 在1x TAE中制备1.5%的200ml琼脂糖 注意:将琼脂糖悬浮液加热至融化,然后将SDS加至0.1%。
    2. 倒入20厘米×24厘米水平板模具。 确保浇铸托盘位于平坦表面上。
      注意:
      1. 消除所有可能的气泡。
      2. 使用水平琼脂糖进行琼脂糖电泳 电泳系统,样品需要长距离运行 以解决高分子量的聚合物。
    3. 安装一个厚梳子(例如,> 2 mm,20-well),每个样品可装入40μl。
    4. 将凝胶浸没在1×TAE-0.1%SDS的水平电泳容器中10℃。
  7. 在10℃下以100V进行电泳7.5小时 注意:为了检测具有高疏水性特征的低聚物,在较低电压50V和室温下,用样品中的1%(终浓度)的Sarkosyl(加样缓冲液中的4%Sarkosyl),在室温下12小时。 运行缓冲液的浓度如前所述是TAE 1x和0.1%SDS。
    注意:电泳时要格外小心,避免电击危险。
  8. 切割调整为琼脂糖凝胶尺寸的PVDF膜,并且将2片与Whatman纸相同尺寸切割。
  9. 将凝胶紧紧地连接到PDVF膜上,并在每一侧添加一块Whatman纸。
    注意:一切都应该用转移缓冲液预湿。 请确保两者之间没有气泡。
  10. 将铸件放入湿转移印迹中,并用缓冲液1x TAE-0.1%SDS覆盖 注意:电泳时要格外小心,避免电击危险。
  11. 在16 V/400 mA和10°C下运行传输15 h
  12. 用适当的抗体进行Western印迹以检测聚合物

食谱

  1. Luria Bertani(LB)培养基
    细菌胰蛋白胨10g/L
    酵母提取物5 g/L
    NaCl 5g/L
  2. 酵母提取物蛋白胨葡萄糖(YPD)
    2%细菌蛋白胨 2%葡萄糖 1%酵母提取物
  3. 裂解缓冲液
    25mM Tris-HCl(pH 6.8)
    250mM NaCl 5 mM EDTA
    10%甘油,补充蛋白酶抑制剂(每10ml一片,每片10ml)
  4. 运行缓冲
    1x TAE(Tris乙酸酯EDTA)
    0.1%SDS
  5. 加载缓冲区(4x)
    0.5%TAE
    5%甘油 2%的sarkosyl
    0.1mg/ml溴酚蓝加蛋白酶抑制剂(每10ml一片,每片1ml)
  6. 传输缓冲区
    1x TAE(Tris Acetate EDTA)
    0.1%SDS

致谢

我们感谢Rafael Giraldo教授的鼓励和持续的支持,以及西班牙MINECO资助(BIO2012-30852和CSD2009-00088)。

参考文献

  1. Coustou,V.,Deleu,C.,Saupe,S.J.and Begueret,J。(1999)。 突变孢子虫的[Het-s]朊病毒类似物的突变分析 。 短的N-末端肽允许朊病毒繁殖。 153(4):1629-1640。
  2. Eaglestone,S.S.,Cox,B.S。和Tuite,M.F。(1999)。 翻译终止效率可通过酿酒酵母进行调节 通过朊病毒介导的机制引起的环境应激。 EMBO J 18(7):1974-1981。
  3. Gasset-Rosa,F.,Coquel,AS,Moreno-Del Alamo,M.,Chen,P.,Song,X.,Serrano,AM,Fernandez-Tresguerres,ME,Moreno-Diaz de la Espina, ,AB和Giraldo,R。(2014)。 在细菌中直接评估朊病毒的繁殖和Hsp70伴侣的表型选择。 Mol Microbiol 91(6):1070-1087
  4. Molina-García,L.和Giraldo,R。(2014)。 RepA-WH1朊病毒变异体之间的聚集相互作用大肠杆菌 l 01527-01514。
  5. Prusiner,S.B。(2012)。 细胞生物学。在神经变性疾病中朊病毒的统一作用。 336(6088):1511-1513。
  6. True,H.L.,Berlin,I.and Lindquist,S.L。(2004)。 翻译的表观遗传调控揭示隐藏的遗传变异以产生复杂的性状。自然 431(7005):184-187。
  7. Wickner,R.B。(1994)。作为改变的URE2蛋白的 [URE3]:酵母菌中朊病毒类似物的证据 264(5158):566-569。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Molina-García, L. and Gasset-Rosa, F. (2014). Semi-denaturing Detergent Agarose Gel Electrophoresis (SDD-AGE). Bio-protocol 4(22): e1297. DOI: 10.21769/BioProtoc.1297.
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