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本实验方案简略版
Jan 2018

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Separation and Visualization of Low Abundant Ubiquitylated Forms
低丰度泛素化形式蛋白的分离和可视化   

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Abstract

In this protocol we describe the separation and visualization of ubiquitylated forms of the yeast mitofusin Fzo1 by Western blot. To this aim, we express HA-tagged Fzo1 in Saccharomyces cerevisiae, break the cells to extract a membrane-enriched fraction, solubilize the membranes using detergent and then specifically immunoprecipitate the tagged protein using anti-HA affinity beads. Subsequently, we separate the higher molecular weight (ubiquitylated) forms of Fzo1 via SDS-PAGE. Finally, immunoblotting and immunodecoration are used to detect the protein and its ubiquitylated forms using an HA-specific antibody. By using this protocol, it is possible to separate and visualize higher molecular weight forms of low abundant proteins such as Fzo1 and detect sharp and distinct bands above the unmodified protein by Western blot.

Keywords: Fzo1 (Fzo1), Ubiquitylation (泛素化), Immunoprecipitation (免疫沉淀), Western blot (免疫印迹), SDS-PAGE (聚丙烯酰胺凝胶电泳)

Background

Immunoprecipitation is a method to precipitate and enrich a protein from an extract by using an antibody specifically binding to it. The enrichment of a protein is of particular importance when analyzing lower abundant modified forms of it, as is usually the case for ubiquitylated forms of a protein. To be able to enrich for the ubiquitylated Fzo1 protein we tagged the protein with a Hemagglutinin (HA) epitope and use anti-HA affinity beads to precipitate this modified protein from the solubilized membrane fraction. This allows for the detection of the less abundant ubiquitylated forms of proteins as for example Fzo1 (Anton et al., 2011 and 2013; Simoes et al., 2018).

Materials and Reagents

  1. Centrifugation tubes (50 ml, 2 ml, 1.5 ml) (Sarstedt, catalog numbers: 62.547.254 [50 ml], 72.695.500 [2 ml], 72.690.001 [1.5 ml])
  2. Nitrocellulose membrane (Amersham, catalog number: 10600001)
  3. Filter papers (Macherey-Nagel, catalog number: 742113)
  4. X-Ray films (Fujifilm, catalog number: 47410 19289)
  5. Transparent plastic foil (Folex, catalog number: 39100440)
  6. Glass beads (Sartorius, catalog number: BBI-8541701)
  7. Yeast Saccharomyces cerevisiae BY4741 isogenic to S288c (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) (Brachmann et al., 1998)
    Note: This protocol is also applicable to other yeasts strains.
  8. D(+)-Glucose monohydrate (Roth, catalog number: 6780.2)
  9.  200 mM PMSF (Serva, catalog number: 32395.03) in Isopropanol (VWR, catalog number: 20842.330)
  10. 50x cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (Roche, catalog number: 05056489001) in MilliQ water
  11. 5% NG310 (Anatrace, catalog number: NG310 1 MG) in TBS buffer
  12. 1 M DTT (MP, catalog number: 100597) in MilliQ water
  13. EZViewTM Red Anti-HA Affinity Gel (Sigma, catalog number: E6779-1ML)
  14. HA primary antibody (clone 3F10) (Roche, catalog number: 11867423001)
  15. Sodium azide (NaN3) (Merck, catalog number: 1.06688.0100)
  16. Rat secondary antibody (Invitrogen, catalog number: 31470)
  17. Tris (Roth, catalog number: 5429.5)
  18. NaCl (Roth, catalog number: 3957.2)
  19. Glycine (PanReac AppliChem, catalog number: 141340.0914)
  20. 37% HCl (VWR, catalog number: 20252.335)
  21. SDS (Roth, catalog number: CN30.3)
  22. Glycerol (Serva, catalog number: 23176.01)
  23. Methanol (Roth, catalog number: 8388.5)
  24. Bromophenol Blue (USB, catalog number: US12370) 
  25. Rotiphorese Gel 30 (Roth, catalog number: 3029.1)
  26. Ponceau S (Serva, catalog number: 33429.02)
  27. Acetic acid (Roth, catalog number: 3738.5)
  28. GE Healthcare AmershamTM ECLTM Prime (Fisher Scientific, catalog number: 10308449)
  29. Nonfat dried milk powder (AppliChem, catalog number: A0830,1000)
  30. Ammonium peroxidisulfate (APS) (Merck, catalog number: 1.01201.0500)
  31. TEMED (Sigma, catalog number: T9281-25ML)
  32. Rotiphorese Gel 30 Acrylamide/Bisacrylamide stock solution (Roth, catalog number: 3029.1)
  33. TBS buffer (see Recipes)
  34. 2x Sample buffer (see Recipes) 
  35. Ponceau S solution (see Recipes)
  36. Tris-Glycine buffer (see Recipes)
  37. Transfer buffer (see Recipes)

Equipment

  1. Rolling pin (e.g., a round pen or a short serological pipette)
  2. Shaking incubator for yeast cultures (Infors-HT, model: Multitron Standard)
  3. Bench-top centrifuge (Eppendorf, model: 5415R)
  4. Vortex (Scientific Industries, model: Vortex-Genie 2)
  5. Glass syringe (Hamilton, e.g., catalog number: 80565) or capillary pipette tips (VWR, catalog number: 53509-015)
  6. Rotator Intelli-Mixer (Elmi, model: RM-2 M)
  7. -20 °C freezer (Liebherr, model: LGUex 1500 MediLine)
  8. Photometer (Hitachi, model: Double Beam Spectrophotometer U-2900)
  9. Thermomixer (Eppendorf, Thermomixer comfort, model: 5355)
  10. Tumbling table (Biometra, model: WT12)
  11. Autoradiography cassette (Amersham Biosciences, HypercassetteTM Autoradiography Cassette model: RPN11642)
  12. Developer machine (AGFA, model: CURIX 60)
  13. Standard SDS-PAGE and western blot equipment
    1. SDS-PAGE equipment, e.g., HoeferTM SE 600 Series
    2. Western Blot equipment, e.g., Peqlab, PerfectBlueTM ‘Semi-Dry’-Blotter, SedecTM

Procedure

  1. Culture conditions
    1. Preculture (Day 1)
      Inoculate cells in 50 ml selective minimal media supplemented with glucose (2%, w/v) and grow overnight to the exponential growth phase (less than 2 OD600 density) at 30 °C by shaking at 160 rpm.
    2. Main culture (Day 2)
      In the morning, inoculate 160 ml selective minimal media supplemented with glucose with a starting OD600 of 0.3 for 4-6 h to the exponential growth phase (less than 2 OD600 density).

  2. Sample preparation
    1. Harvest 160 OD600 of these exponentially growing cells per condition using 50 ml conical tubes by centrifuging (5 min, 4,100 x g, RT). Discard supernatant.
    2. Resuspend the pellets in 1 ml TBS buffer and transfer the suspension to two 2 ml round-bottom centrifugation tubes and centrifuge (1 min, 16,100 x g, 4 °C). Discard supernatant. 

    Note: Proceed with all following steps on ice.
    1. Resuspend pellets in 300 µl TBS buffer each. Add 750 mg glass beads, 10 µl 0.2 M PMSF and 20 µl 50x cOmpleteTM EDTA-free protease inhibitor to each suspension.
    2. Disrupt the cells by vigorous vortexing using the highest setting (Vortex) (3x 30 s vortexing, 30 s on ice, alternating).
    3. Pool the two suspensions of corresponding conditions.
    4. Add 400 µl TBS buffer.
    5. Centrifuge mildly (2 min, 1,500 x g, 4 °C). 
    6. Transfer the supernatant to a new 1.5 ml centrifugation tube.
    7. Centrifuge (15 min, 16,100 x g, 4 °C). Discard supernatant. 
    8. Resuspend the pellet (= crude membranes) in cold 0.2% NG310 in TBS buffer.
    9. Solubilize the membranes by continuously rotating the tube (1 h, 10 rpm, 4 °C) using a rotator.
    10. During solubilization, prepare tubes.
      1. Input control (In): 20 µl 2x Sample buffer with freshly added 0.1 M DTT.
      2. Immunoprecipitation (IP): 500 µl 0.2% NG310 in TBS buffer + 23 µl Anti-HA Affinity Gel (wash the Anti-HA Affinity Gel once with TBS buffer before adding it).
    11. After solubilization, centrifuge (5 min, 16,100 x g, 4 °C).
    12. Take 20 µl of the supernatant as input control (= 4%), mix it with the 2x sample buffer in the prepared tube (In) and freeze until use at -20 °C.
    13. Add the remaining supernatant to the prepared IP tube and rotate the tube (overnight, 10 rpm, 4 °C) using a rotator.
    14. (Day 3) Centrifuge IP tube (30 s, 400 x g, 4 °C).
    15. Remove supernatant completely
      The Anti-HA Affinity Gel consists of small gelatinous beads. As not to aspirate the beads use a syringe with a small opening or thin capillary pipette tips (refer to Equipment section for details).
    16. Wash the Anti-HA Affinity Gel with 0.2% NG310 in TBS buffer.
    17. Repeat washing 3 x (Steps B16-B18).
    18. Add 60 µl 2x sample buffer with freshly added 0.1 M DTT.
    19. Thaw the input control on ice.
    20. Heat samples using a thermomixer (20 min, 1,400 rpm, 45 °C).
    21. Centrifuge (30 s, 1,500 x g, RT).

  3. SDS-PAGE and Immunoblotting
    1. Separate samples using SDS-PAGE
      1. Assemble gel equipment according to manufacturer’s instructions (see Equipment section for details).
      2. Prepare separation and stacking gel mixtures (do not add APS and TEMED yet as it will start the polymerization process).
        For 2 gels:

        1 30% Acrylamide with 0.8% Bisacrylamide (37.5:1)

      3. Add APS and TEMED to the separation gel mixture and mix well. For each gel, with a pipette, rapidly pour 9 ml of the separation gel mix between the two glass plates and overlay each with 1 ml of isopropanol to even out the surface. Keep the remaining gel mixture and use it to control the polymerization status. After polymerization, decant the isopropanol and wash with water carefully. Remove excess water with filter paper.
      4. Put in the combs, leaving 3-5 mm distance from the separation gel. Add APS and TEMED to the stacking gel mixture and mix well. Rapidly pour the stacking gel mixture on top, making sure that no bubbles appear in the stacking gel. Keep the remaining gel mixture and use it to control the polymerization status. After polymerization, remove the comb and fill the slots with 1x Tris-Glycine buffer. Load samples (e.g., 4% Input control and 100% eluate) and a pre-stained protein marker of choice (e.g., NEB, P7712).
      5. Fill SDS-PAGE tank with Tris-Glycine buffer to the filling line (anode). Insert gels into the tank as per manufacturer’s instructions (make sure that the bottom parts of the gels are in contact with the anode buffer). Fill cathode reservoir with enough Tris-Glycine buffer to cover the cathode.
      6. Run Gels at 150-200 V until the protein of interest reaches the bottom of the gel (the protein itself will not be visible on the gel, thus refer to the pre-stained protein marker to estimate when to stop the gel.)
        Note: To make sure that the higher molecular weight ubiquitylation bands will be separated efficiently the unmodified protein needs to run as far as possible.
      7. Disassemble the gels by removing them from the tanks and by taking the glass plates apart carefully with the plastic wedge coming with the SDS-PAGE electrophoresis unit. Cut off the stacking gel with the plastic wedge.
    2. Transfer the proteins from the acrylamide gel to a membrane by semi-dry western blotting
      1. Soak six filter papers, one nitrocellulose membrane and the separation gel briefly in transfer buffer.
      2. Assemble western blotting sandwich as described from plus to minus pole: three filter papers, one nitrocellulose membrane, gel, three filter papers. Between each layer, carefully remove bubbles by rolling over the sandwich with a rolling pin (e.g., a round pen or a short serological pipette).
      3. Run the transfer for 2 h for 2 mA per cm2 gel/membrane (i.e., for one 7 x 15 cm gel: 2 h, 200 mA).
    3. Wash the membrane with desalted water, stain the proteins with Ponceau S solution and scan the membranes (only the input control will show a staining), destain using TBS buffer, block the membranes at room temperature for 30 min in 5% milk in TBS buffer, all steps while shaking at 40 rpm using a tumbling table.
    4. Perform immunodecoration to detect the HA epitope (Steps a-d require shaking at 40 rpm on a tumbling table). A representative image is shown in Figure 1.
      1. Replace the blocking solution with the primary antibody solution: 1:1,000 anti-HA in 5% milk in TBS buffer and 0.02% NaN3 and incubate overnight at 4 °C.
      2. Remove the primary antibody solution (can be stored at -20 °C if desired) and wash 3 x using TBS buffer at room temperature for 10 min each.
      3. Replace the TBS buffer with the secondary antibody solution: 1:5,000 anti-rat in 5% milk in TBS buffer for 2 h incubation at room temperature.
      4. Wash 3 x for 10 min using TBS buffer at room temperature. Tilt the membrane after the last wash to remove excess liquid.
      5. Detect the signal.
        1. Place membrane in a flat container.
        2. Mix equal amounts of solutions A and B (e.g., 1 ml total volume) of ECL and repeatedly spread over the membrane (for 30 s).
        3. Tilt the membrane to remove excess liquid, place it in an autoradiography cassette and cover with a transparent plastic foil. Proceed quickly now as the signal may rapidly fade and also because big amounts of protein may burn the membranes irreparably.
          Note: For big amounts of protein as here, the membrane will most likely burn at the position of the unmodified protein, therefore the development of the membrane will not be repeatable without quality loss.
        4. In a film developing room (only red light!), place unexposed X-Ray films on the membrane for varying durations, starting with short exposures (e.g., from 1 s to 30 min). Place and remove them in a fast movement without sliding across the membrane to get a sharp signal.
        5. Use a developer machine to develop the films. 


      Figure 1. Visualization of low abundant ubiquitylated forms of Fzo1 by immunoprecipitation and western blot analysis

Recipes

  1. TBS buffer
    50 mM Tris
    150 mM NaCl
    Adjust the pH to 7.5 using HCl 37%
  2. 2x Sample buffer
    4% SDS
    20% Glycerol
    135 mM Tris-HCl (pH 6.8)
    0.1% Bromophenol Blue
    Store at RT. Add 100 mM DTT freshly before use
  3. Ponceau S solution
    0.1% Ponceau S
    5% Acetic acid
  4. Tris-Glycine buffer
    25 mM Tris
    200 mM Glycine
    3.5 mM SDS
  5. Transfer buffer
    200 mM Glycine
    25 mM Tris
    20% (v/v) Methanol
    0.02% SDS
    Adjust pH to 8.5 using HCl 37%

Acknowledgments

Funding: Deutsche Forschungsgemeinschaft (ES338/3-1); Universität zu Köln (German Excellence Initiative and Faculty of Mathematics and Natural Sciences); Deutsche Forschungsgemeinschaft (SFB635); Deutsche Forschungsgemeinschaft (CRC1218TPA03).

Competing interests

The authors declare they have no conflict of interest or competing interests.

References

  1. Anton, F., Dittmar, G., Langer, T. and Escobar-Henriques, M. (2013). Two deubiquitylases act on mitofusin and regulate mitochondrial fusion along independent pathways. Mol Cell 49(3): 487-498.
  2. Anton, F., Fres, J. M., Schauss, A., Pinson, B., Praefcke, G. J., Langer, T. and Escobar-Henriques, M. (2011). Ugo1 and Mdm30 act sequentially during Fzo1-mediated mitochondrial outer membrane fusion. J Cell Sci 124(Pt 7): 1126-1135.
  3. Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P. and Boeke, J. D. (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14(2): 115-132.
  4. Simoes, T., Schuster, R., den Brave, F. and Escobar-Henriques, M. (2018). Cdc48 regulates a deubiquitylase cascade critical for mitochondrial fusion. Elife 7: e30015.

简介

在该方案中,我们描述了通过蛋白质印迹分离和可视化酵母mitofusin Fzo1的泛素化形式。 为此目的,我们在酿酒酵母中表达HA标记的Fzo1,打破细胞以提取富含膜的部分,使用去污剂溶解膜,然后使用抗HA亲和珠特异性地免疫沉淀标记的蛋白质。 随后,我们通过SDS-PAGE分离较高分子量(泛素化)形式的Fzo1。 最后,使用免疫印迹和免疫装饰使用HA特异性抗体检测蛋白质及其泛素化形式。 通过使用该方案,可以分离和可视化较高分子量形式的低丰度蛋白质,例如Fzo1,并通过蛋白质印迹检测未修饰蛋白质上方的清晰和明显的条带。

【背景】免疫沉淀是通过使用与其特异性结合的抗体从提取物中沉淀和富集蛋白质的方法。 当分析其较低丰度的修饰形式时,蛋白质的富集是特别重要的,这通常是泛素化形式的蛋白质的情况。 为了能够富集泛素化的Fzo1蛋白,我们用血凝素(HA)表位标记蛋白质,并使用抗HA亲和珠从溶解的膜部分中沉淀出这种修饰的蛋白质。 这允许检测不太丰富的泛素化形式的蛋白质,例如Fzo1(Anton 等人,2011和2013; Simoes 等人,2018)。

关键字:Fzo1, 泛素化, 免疫沉淀, 免疫印迹, 聚丙烯酰胺凝胶电泳

材料和试剂

  1. 离心管(50ml,2ml,1.5ml)(Sarstedt,目录号:62.547.254 [50ml],72.695.500 [2ml],72.690.001 [1.5ml])
  2. 硝酸纤维素膜(Amersham,目录号:10600001)
  3. 滤纸(Macherey-Nagel,目录号:742113)
  4. X光片(Fujifilm,目录号:47410 19289)
  5. 透明塑料薄膜(Folex,目录号:39100440)
  6. 玻璃珠(赛多利斯,产品目录号:BBI-8541701)
  7. 酵母 Saccharomyces cerevisiae BY4741与S288c同基因(MATa his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 )(Brachmann et al。,1998)
    注意:该方案也适用于其他酵母菌株。
  8. D(+) - 葡萄糖一水合物(Roth,目录号:6780.2)
  9.   200mM PMSF(Serva,目录号:32395.03)在异丙醇中(VWR,目录号:20842.330)
  10. 在MilliQ水中50x cOmplete TM,不含EDTA的蛋白酶抑制剂混合物(Roche,目录号:05056489001)
  11. TBS缓冲液中的5%NG310(Anatrace,目录号:NG310 1 MG)
  12. MilliQ水中的1 M DTT(MP,目录号:100597)
  13. EZView TM红色抗HA亲和凝胶(Sigma,目录号:E6779-1ML)
  14. HA一抗(克隆3F10)(罗氏,目录号:11867423001)
  15. 叠氮化钠(NaN 3)(默克,目录号:1.06688.0100)
  16. 大鼠二抗(Invitrogen,目录编号:31470)
  17. Tris(罗斯,目录号:5429.5)
  18. NaCl(Roth,目录号:3957.2)
  19. 甘氨酸(PanReac AppliChem,目录号:141340.0914)
  20. 37%HCl(VWR,目录号:20252.335)
  21. SDS(Roth,目录号:CN30.3)
  22. 甘油(Serva,目录号:23176.01)
  23. 甲醇(罗斯,目录号:8388.5)
  24. 溴酚蓝(USB,目录号:US12370) 
  25. Rotiphorese Gel 30(罗斯,目录号:3029.1)
  26. Ponceau S(Serva,目录号:33429.02)
  27. 乙酸(罗斯,目录号:3738.5)
  28. GE Healthcare Amersham TM ECL TM Prime(Fisher Scientific,目录号:10308449)
  29. 脱脂奶粉(AppliChem,目录号:A0830,1000)
  30. 过氧化硫酸铵(APS)(默克,目录号:1.01201.0500)
  31. TEMED(Sigma,目录号:T9281-25ML)
  32. Rotiphorese Gel 30丙烯酰胺/双丙烯酰胺原液(Roth,目录号:3029.1)
  33. TBS缓冲区(见食谱)
  34. 2x样品缓冲液(参见食谱) 
  35. Ponceau S解决方案(见食谱)
  36. Tris-Glycine缓冲液(见食谱)
  37. 转移缓冲区(见食谱)

设备

  1. 擀面杖(例如,圆形笔或短血清移液管)
  2. 摇动酵母培养箱(Infors-HT,型号:Multitron Standard)
  3. 台式离心机(Eppendorf,型号:5415R)
  4. Vortex(科学工业,型号:Vortex-Genie 2)
  5. 玻璃注射器(Hamilton,例如,目录号:80565)或毛细管移液器吸头(VWR,目录号:53509-015)
  6. Rotator Intelli-Mixer(Elmi,型号:RM-2 M)
  7. -20°C冰柜(利勃海尔,型号:LGUex 1500 MediLine)
  8. 光度计(日立,型号:双光束分光光度计U-2900)
  9. Thermomixer(Eppendorf,Thermomixer comfort,型号:5355)
  10. 翻滚台(Biometra,型号:WT12)
  11. 放射自显影盒(Amersham Biosciences,Hypercassette TM放射自显影盒型号:RPN11642)
  12. 开发者机器(AGFA,型号:CURIX 60)
  13. 标准SDS-PAGE和western blot设备
    1. SDS-PAGE设备,例如,Hoefer TM SE 600系列
    2. Western Blot设备,例如,Peqlab,PerfectBlue TM'Semi-Dry'-Blotter,Sedec TM

程序

  1. 文化条件
    1. 预培养(第1天)
      在补充有葡萄糖(2%,w / v)的50ml选择性基本培养基中接种细胞,并在30℃下通过摇动在160℃下生长过夜至指数生长期(小于2OD 600密度)。转。
    2. 主要文化(第2天)
      早上,接种160 ml选择性基本培养基,补充葡萄糖,起始OD 600 0.3,持续4-6小时至指数生长期(小于2 OD 600密度)。

  2. 样品制备
    1. 通过离心(5分钟,4,100 x g,RT),使用50ml锥形管每种条件收获160个OD 600这些指数生长的细胞。丢弃上清液。
    2. 将沉淀重悬于1ml TBS缓冲液中,并将悬浮液转移至两个2ml圆底离心管中并离心(1分钟,16,100 x g ,4℃)。丢弃上清液。 

    注意:在冰上继续执行以下所有步骤。
    1. 将沉淀重悬于300μlTBS缓冲液中。向每种悬浮液中加入750mg玻璃珠,10μl0.2MPMSF和20μl50x完全 TM不含EDTA的蛋白酶抑制剂。
    2. 使用最高设置(涡旋)(3x 30秒涡旋,30秒冰上,交替)通过剧烈涡旋破坏细胞。
    3. 汇集相应条件的两个暂停。
    4. 加入400μlTBS缓冲液。
    5. 温和地离心(2分钟,1,500 x g ,4°C)。 
    6. 将上清液转移到新的1.5ml离心管中。
    7. 离心(15分钟,16,100 x g ,4℃)。丢弃上清液。 
    8. 将沉淀(=粗膜)重悬于TBS缓冲液中的冷0.2%NG310中。
    9. 通过使用旋转器连续旋转管(1小时,10rpm,4℃)来溶解膜。
    10. 在溶解过程中,准备管。
      1. 输入对照(In):20μl2x样品缓冲液,新加入0.1M DTT。
      2. 免疫沉淀(IP):在TBS缓冲液中的500μl0.2%NG310 +23μl抗HA亲和凝胶(在添加之前用TBS缓冲液洗涤抗HA亲和凝胶一次)。
    11. 溶解后,离心(5分钟,16,100 x g ,4℃)。
    12. 取20μl上清液作为输入对照(= 4%),将其与制备的管(In)中的2x样品缓冲液混合并冷冻直至-20°C使用。
    13. 将剩余的上清液加入到制备的IP管中并使用旋转器旋转管(过夜,10rpm,4℃)。
    14. (第3天)离心IP管(30秒,400 x g ,4℃)。
    15. 完全去除上清液
      抗HA亲和凝胶由小凝胶珠组成。为了不吸出珠子,请使用带有小开口或薄毛细管移液器吸头的注射器(有关详细信息,请参阅设备部分)。
    16. 用含有0.2%NG310的TBS缓冲液洗涤抗HA亲和凝胶。
    17. 重复洗涤3次(步骤B16-B18)。
    18. 加入60μl2x样品缓冲液,加入新鲜的0.1M DTT。
    19. 在冰上解冻输入控制。
    20. 使用恒温混匀器加热样品(20分钟,1,400转/分钟,45°C)。
    21. 离心(30秒,1,500 x g ,RT)。

  3. SDS-PAGE和免疫印迹
    1. 使用SDS-PAGE分离样品
      1. 根据制造商的说明组装凝胶设备(有关详细信息,请参阅设备部分)。
      2. 准备分离和堆积凝胶混合物(不要添加APS和TEMED,因为它将开始聚合过程)。
        对于2凝胶:

        1 30%丙烯酰胺与0.8%双丙烯酰胺(37.5:1)

      3. 将APS和TEMED加入分离凝胶混合物中并充分混合。对于每种凝胶,用移液管在两块玻璃板之间快速倒入9ml分离凝胶混合物,并用1ml异丙醇覆盖,使表面均匀。保留剩余的凝胶混合物并用它来控制聚合状态。聚合后,倾倒异丙醇并小心地用水洗涤。用滤纸除去多余的水分。
      4. 放入梳子,离开分离凝胶3-5毫米。将APS和TEMED加入到堆积凝胶混合物中并充分混合。将堆积凝胶混合物快速倒在上面,确保堆积凝胶中没有气泡。保留剩余的凝胶混合物并用它来控制聚合状态。聚合后,取出梳子并用1x Tris-甘氨酸缓冲液填充槽。加载样品(例如,4%输入对照和100%洗脱液)和预先选择的蛋白质标记物(例如,NEB,P7712)。
      5. 将具有Tris-甘氨酸缓冲液的SDS-PAGE槽填充至填充线(阳极)。按照制造商的说明将凝胶插入罐中(确保凝胶的底部与阳极缓冲液接触)。用足够的Tris-甘氨酸缓冲液填充阴极贮存器以覆盖阴极。
      6. 在150-200 V下运行凝胶,直到感兴趣的蛋白质到达凝胶底部(蛋白质本身在凝胶上不可见,因此参考预先染色的蛋白质标记物来估计何时停止凝胶。)
        注意:为了确保更高分子量的泛素化条带能够有效分离,未修饰的蛋白质需要尽可能地运行。
      7. 将凝胶从罐中取出并通过SDS-PAGE电泳装置附带的塑料楔小心地将玻璃板分开来拆卸凝胶。用塑料楔子切掉堆叠凝胶。
    2. 通过半干免疫印迹将蛋白质从丙烯酰胺凝胶转移到膜上
      1. 在转移缓冲液中简单地浸泡六个滤纸,一个硝酸纤维素膜和分离凝胶。
      2. 如上所述组装蛋白质印迹夹心从正极到负极:三个滤纸,一个硝酸纤维素膜,凝胶,三个滤纸。在每层之间,用擀面杖(例如,圆形笔或短血清移液管)在夹层上滚动,小心地去除气泡。
      3. 进行转移2小时,每cm 22μl 2凝胶/膜(即,对于一个7×15cm凝胶:2小时,200mA)。
    3. 用脱盐水洗涤膜,用Ponceau S溶液染色蛋白质并扫描膜(仅输入对照将显示染色),使用TBS缓冲液脱色,在室温下在TBS缓冲液中的5%牛奶中封闭膜30分钟,使用翻滚台以40转/分钟的速度摇晃所有步骤。
    4. 进行免疫装饰以检测HA表位(步骤a-d需要在翻滚台上以40rpm振荡)。代表性的图像如图1所示。
      1. 用一抗溶液替换封闭溶液:在TBS缓冲液和0.02%NaN 3中的5%牛奶中1:1,000抗HA,并在4℃下孵育过夜。
      2. 除去一抗溶液(如果需要可以在-20℃下保存)并在室温下使用TBS缓冲液洗涤3次,每次10分钟。
      3. 用二抗溶液替换TBS缓冲液:在5%牛奶中在TBS缓冲液中1:5,000抗大鼠在室温下孵育2小时。
      4. 在室温下使用TBS缓冲液洗涤3次,每次10分钟。最后一次洗涤后将膜倾斜以除去多余的液体。
      5. 检测信号。
        1. 将膜放入扁平容器中。
        2. 混合等量的溶液A和B(例如,1ml总体积)的ECL并重复铺展在膜上(30秒)。
        3. 倾斜膜以去除多余的液体,将其放入放射自显影盒中并用透明塑料箔覆盖。现在快速进行,因为信号可能会迅速消退,同时因为大量蛋白质可能会不可挽回地燃烧膜。
          注意:对于这里的大量蛋白质,膜很可能在未修饰蛋白质的位置燃烧,因此如果没有质量损失,膜的发展将不可重复。
        4. 在胶片显影室(仅红光!)中,将未曝光的X射线胶片放置在胶片上不同的持续时间,从短曝光开始(例如,从1秒到30分钟)。快速放置并移除它们,不要在膜上滑动以获得清晰的信号。
        5. 使用开发者机器开发电影。 


      图1.通过免疫沉淀和蛋白质印迹分析可视化低丰度泛素化形式的Fzo1

食谱

  1. TBS缓冲区
    50 mM Tris
    150 mM NaCl
    使用HCl 37%将pH调节至7.5
  2. 2x样品缓冲液
    4%SDS
    20%甘油
    135mM Tris-HCl(pH6.8)
    0.1%溴酚蓝
    在RT存储。使用前新鲜加入100 mM DTT
  3. Ponceau S解决方案
    0.1%Ponceau S
    5%乙酸
  4. Tris-甘氨酸缓冲液
    25 mM Tris
    200 mM甘氨酸
    3.5mM SDS
  5. 转移缓冲区
    200 mM甘氨酸
    25 mM Tris
    20%(v / v)甲醇
    0.02%SDS
    使用37%HCl将pH调节至8.5

致谢

资金来源:Deutsche Forschungsgemeinschaft(ES338 / 3-1); UniversitätzuKöln(德国卓越计划和数学与自然科学学院); Deutsche Forschungsgemeinschaft(SFB635); Deutsche Forschungsgemeinschaft(CRC1218TPA03)。

利益争夺

作者声明他们没有利益冲突或竞争利益。

参考

  1. Anton,F.,Dittmar,G.,Langer,T。和Escobar-Henriques,M。(2013)。 两种deubiquitylases作用于mitofusin并调节独立通路的线粒体融合。 Mol Cell 49(3):487-498。
  2. Anton,F.,Fres,J.M.,Schauss,A.,Pinson,B.,Praefcke,G.J.,Langer,T。和Escobar-Henriques,M。(2011)。 Ugo1和Mdm30在Fzo1介导的线粒体外膜融合过程中依次起作用。 J Cell Sci 124(Pt 7):1126-1135。
  3. Brachmann,C.B.,Davies,A.,Cost,G.J.,Caputo,E.,Li,J.,Hieter,P。和Boeke,J.D。(1998)。 来自酿酒酵母S288C的设计者缺失菌株:一组有用的PCR介导基因菌株和质粒中断和其他应用程序。 Yeast 14(2):115-132。
  4. Simoes,T.,Schuster,R.,den Brave,F。和Escobar-Henriques,M。(2018)。 Cdc48调节对线粒体融合至关重要的deubiquitylase级联。 Elife 7:e30015。
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Copyright Schuster et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Schuster, R., Simões, T., den Brave, F. and Escobar-Henriques, M. (2018). Separation and Visualization of Low Abundant Ubiquitylated Forms. Bio-protocol 8(22): e3081. DOI: 10.21769/BioProtoc.3081.
  2. Simoes, T., Schuster, R., den Brave, F. and Escobar-Henriques, M. (2018). Cdc48 regulates a deubiquitylase cascade critical for mitochondrial fusion. Elife 7: e30015.
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