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Immunoprecipitation of Proteins in Caenorhabditis elegans
免疫沉淀法检测秀丽隐杆线虫蛋白质   

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Abstract

Immunoprecipitation (IP) is a biochemical technique to precipitate a protein out of solution using an antigen that can specifically bind to that protein. IP can be performed to isolate and concentrate one particular protein from a sample of thousands of different proteins. IP is also readily performed to pull down interacting proteins of complexes out of solution. This protocol outlines the methods used to IP proteins in whole worm lysates and their preparation for detection on Western blots using denaturing conditions.

Materials and Reagents

  1. Transgenic Caenohabditis elegans (C. elegans) strain with over-expressed protein of interest
    Note: Depending on protein expression and specificity of antibody, this protocol can also be used to immunoprecipitate endogenous proteins.
  2. OP50 Escherichia coli (E. coli) (C. elegans Genetic Center)
  3. Trizma hydrochloride (Sigma-Aldrich, catalog number: T5941 )
  4. Trizma base (Sigma-Aldrich, catalog number: T1503 )
  5. Tris base (BioShop, catalog number: TRS001 )
  6. Bacto-tryptone (BD, catalog number: 211705 )
  7. NaCl (BioShop, catalog number: SOD002 )
  8. Cholesterol (95%) (Sigma-Aldrich, catalog number: C8503 )
  9. Agar (Sigma-Aldrich, catalog number: A1296 )
  10. Na2HPO4 (Sigma-Aldrich, catalog number: S0876 )
  11. KH2PO4 (Sigma-Aldrich, catalog number: P0662 )
  12. MgSO4·7H2O (BioShop, catalog number: MAG521 )
  13. EDTA (BioBasic, catalog number: EB0185 )
  14. NP-40 (BioShop, catalog number: NON.505 )
  15. PMSF (Roche, catalog number: 10837091001 )
  16. Na3VO4 (Sigma-Aldrich, catalog number: S6508 )
  17. Pepstatin-A (Sigma-Aldrich, catalog number: P4265 )
  18. NaF (Sigma-Aldrich, catalog number: S7920 )
  19. cOmplete protease inhibitor cocktail tablets (Roche, catalog number: 4693159001 )
  20. SDS (Caledon, catalog number: 7771 )
  21. Glycerol (BioShop, catalog number: GLY001 )
  22. β-mercaptoethanol (Sigma-Aldrich, catalog number: M7154 )
  23. Bromophenol blue (Sigma-Aldrich, catalog number: B5525 )
  24. BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23225 )
  25. Siliconized low-binding tips 200-1,000 μl (Denville Scientific Inc., catalog number: P3193-S )
  26. Siliconized low-binding tips 200 μl (Denville Scientific Inc., catalog number: P3010-S )
  27. Protein A/G PLUS-Agarose non-conjugated beads (Santa Cruz Biotechnology, catalog number: SC-2003 )
  28. EZview Red ANTI-FLAG M2 Affinity Gel conjugated beads (Sigma-Aldrich, catalog number: F2426 )
  29. GFP polyclonal antibody, rabbit (Genscript, catalog number: A01388 )
  30. GFP monoclonal antibody (B-2) (mouse) (Santa Cruz Biotechnology, catalog number: SC-9996 )
  31. Donkey anti-rabbit IgG-HRP (Santa Cruz Biotechnology, catalog number: SC-2313 )
  32. Goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, catalog number: SC-2005 )
  33. MYOB dry mix (see Recipes)
  34. MYOB plates (see Recipes)
  35. M9 buffer (see Recipes)
  36. Lysis buffer (see Recipes)
  37. SDS-PAGE sample buffer (see Recipes)

Equipment

  1. Refrigerated microcentrifuge (Eppendorf, model: 5417R )
  2. Large centrifuge (Eppendorf, model: 5810R )
  3. Rotisserie agitator (Barnstead Thermolyne LabQuake, model: C415110 )
  4. Sonicator (Branson Sonifier, model: 450 )

Procedure

  1. Collection of worms
    1. Propagate desired strain of worms using the OP50 E. coli seeded on MYOB plates. 15 fully confluent (not starved) 10 cm plates of worms gives enough total protein for two IPs (10-25 mg).
    2. Collect all worms off of plates with 15 ml of M9 buffer into a 15 ml conical tube.
    3. Spin down worms at 2,000 rpm for 1 min and replace supernatant with fresh M9 buffer.
      Note: Repeat this step until worms are relatively clean of bacteria (clear supernatant).
    4. Resuspend worms by gentle inversion in 15 ml PBS.
    5. Spin down worms and remove as much supernatant as possible.
    6. Transfer up to 500 μl of packed worms using siliconized tips into a microcentrifuge tube.
      Note: Siliconized tips are recommended to prevent adherence of worms.
    7. Add up to 1 ml of ice cold lysis buffer (2x worm volume) and incubate on ice for 30 min.
      Note: Worms can be flash frozen using liquid nitrogen and stored at -80 °C up to one week. For best results, continue with lysis immediately.

  2. Lysis of worms
    1. If performing lysis on same day, flash freeze worms in liquid nitrogen for 10 sec. If performing lysis on previously stored worm samples, continue to step B2.
    2. Thaw frozen worms in room temperature water until a third of the frozen sample has melted.
    3. Insert tip of sonicator halfway into sample and sonicate for 5-7 sec using 8 W power. Immediately flash freeze samples for 10 sec in liquid nitrogen.
      Note: During sonication, do not allow samples to completely thaw during this process. It is important to keep the sample cold at all times to avoid protein degradation. Incubating samples on ice during sonication is optimal. One alternative that has been previously reported (Ding et al., 2005) is to use a pestle and mortar to pulverize the worm pellet in the presence of liquid nitrogen to disrupt the cuticle, which may help increase protein yield and preserve the integrity of proteins.
    4. Repeat steps B2-3 two times. After final flash freeze, let samples thaw in room temperature water until half melted.
    5. Rotate samples at 4 °C by end-over-end agitation for 30 min.
    6. Spin down samples at 13,000 rpm for 30 min at 4 °C.
    7. Transfer supernatant (solubilized proteins) into a new microcentrifuge tube and measure protein concentration using a standard kit (e.g. BCA protein assay kit).

  3. Immunoprecipitation
    1. At least 4 mg of total protein was used for each IP. 8-10 mg of protein is recommended. Adjust the volume of protein sample to 1 ml using ice cold lysis buffer.
      Note: It is not recommended to store samples for freezing at this step.
    2. Equilibrate agarose beads by washing in lysis buffer three times using 2x volume of bead slurry.
      1. If using antibody-conjugated beads, add 20-30 μl of equilibrated beads to protein sample and incubate at 4 °C with agitation for 2-4 h.
        Note: Incubation of beads beyond 4 h did not change immunoprecipitation efficacy in our hands.
      2. If using non-conjugated beads, first add antibody to sample and incubate at 4 °C with agitation for 1 h. Then add 20-30 μl of equilibrated beads and further incubate for another 1-3 h.
        Note: We have had experience using both antibody-conjugated agarose beads and non-conjugated agarose beads. We did not detect any notable differences between IP efficiency.
    3. After incubation, wash beads 5 times using 1 ml ice cold lysis buffer, allowing samples to incubate with agitation at 4 °C for 2-5 min between washes.
    4. Elute proteins with 50-100 μl of SDS-PAGE sample buffer and boiling for 5 min, analyze by Western blot.
      Notes:
      1. When performing Western blot, another 5-10 minutes of boiling is recommended to elute proteins from beads.
      2. For several examples of Western blots whose samples were prepared using this protocol, please refer to Chan et al. (2014). The specificity of the immunoprecipitation will depend upon the degree of specificity of the antibody being used. For best results, pre-clear the lysates with an irrelevant antibody to improve signal-to-noise ratio. Regardless, several different types of controls can be performed to evaluate the specificity of the immunoprecipitation, including performing the immunoprecipitation with a sample that does not contain the epitope of interest (Chan et al., 2014), or including a mock immunoprecipitation control, whereby antibody is not added to sample before the immunoprecipitation.

Recipes

Note: Recipes for MYOB and M9 were taken from Burns et al. (2006).

  1. MYOB dry mix (for 370 g)
    27.5 g trizma HCl
    12 g trizma base
    230 g Bacto-tryptone
    100 g NaCl
    0.4 g cholesterol (95%)
  2. MYOB plates (for 1 L)
    7.4 g MYOB dry mix
    24 g agar
    Add up to 1 L ddH2O
    Autoclave and pour 35 ml per 10 cm plate
  3. M9 buffer (for 1 L)
    6 g Na2HPO4
    3 g KH2PO4
    5 g NaCl
    Add up to 1 L ddH2O
    After cooling to room temperature, add 2 ml of 1 M MgSO4·7H2O
  4. Lysis buffer
    25 mM Tris-HCl (pH 7.5)
    100 mM NaCl
    1 mM EDTA
    0.5% NP-40
    1 mM PMSF
    1 mM Na3VO4
    1 μg/ml Pepstatin-A
    10 mM NaF
    1 tablet/50 ml buffer cOmplete protease inhibitor cocktail tablets
    ddH2O to desired final volume
  5. SDS-PAGE sample buffer
    100 mM Tris-HCl (pH 6.8)
    2% SDS
    20% glycerol
    4% β-mercaptoethanol
    0.02% bromophenol blue

Acknowledgments

This work was supported by grants from the Canadian Cancer Society Research Institute (# 020511) and Canadian Institutes of Health Research (#258898) to PJR.

References

  1. Burns, A. R., Kwok, T. C., Howard, A., Houston, E., Johanson, K., Chan, A., Cutler, S. R., McCourt, P. and Roy, P. J. (2006). High-throughput screening of small molecules for bioactivity and target identification in Caenorhabditis elegans. Nat Protoc 1(4): 1906-1914.
  2. Chan, K. K., Seetharaman, A., Bagg, R., Selman, G., Zhang, Y., Kim, J. and Roy, P. J. (2014). EVA-1 functions as an UNC-40 Co-receptor to enhance attraction to the MADD-4 guidance cue in Caenorhabditis elegans. PLoS Genet 10(8): e1004521.
  3. Ding, L., Spencer, A., Morita, K. and Han, M. (2005). The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans. Mol Cell 19(4): 437-447.
  4. Hall, R. A. (2005). Co-Immunoprecipitation as a strategy to evaluate receptor-receptor or receptor-protein interactions. In: George, S. R., and Dowd, B. F. (eds). G Protein-Coupled Receptor-Protein Interactions. John Wiley & Sons, Inc., 165-178.

简介

免疫沉淀(IP)是使用可以特异性结合该蛋白质的抗原将蛋白质从溶液中沉淀出来的生物化学技术。 IP可以从数千种不同蛋白质的样品中分离和浓缩一种特定蛋白质。 IP也容易进行以从溶液中取出复合物的相互作用蛋白。 该协议概述了用于IP蛋白在整个蠕虫裂解物中的方法及其用于使用变性条件在蛋白质印迹上检测的方法。

材料和试剂

  1. 具有过表达的目标蛋白质的转基因秀丽隐杆线虫( C. elegans )菌株
    注意:根据蛋白质表达和抗体的特异性,该方案也可用于免疫沉淀内源性蛋白质。
  2. OP50大肠杆菌(大肠杆菌)( C. elegans 遗传中心)
  3. 盐酸三甲胺(Sigma-Aldrich,目录号:T5941)
  4. Trizma碱(Sigma-Aldrich,目录号:T1503)
  5. Tris碱(BioShop,目录号:TRS001)
  6. 细菌胰蛋白胨(BD,目录号:211705)
  7. NaCl(BioShop,目录号:SOD002)
  8. 胆固醇(95%)(Sigma-Aldrich,目录号:C8503)
  9. 琼脂(Sigma-Aldrich,目录号:A1296)
  10. Na 2 HPO 4(Sigma-Aldrich,目录号:S0876)
  11. KH sub 2 PO 4(Sigma-Aldrich,目录号:P0662)
  12. MgSO 4·7H 2 O(BioShop,目录号:MAG521)
  13. EDTA(BioBasic,目录号:EB0185)
  14. NP-40(BioShop,目录号:NON.505)
  15. PMSF(Roche,目录号:10837091001)
  16. (Sigma-Aldrich,目录号:S6508)
  17. 胃蛋白酶抑制剂A(Sigma-Aldrich,目录号:P4265)
  18. NaF(Sigma-Aldrich,目录号:S7920)
  19. cOmplete蛋白酶抑制剂混合物片剂(Roche,目录号:4693159001)
  20. SDS(Caledon,目录号:7771)
  21. 甘油(BioShop,目录号:GLY001)
  22. β-巯基乙醇(Sigma-Aldrich,目录号:M7154)
  23. 溴酚蓝(Sigma-Aldrich,目录号:B5525)
  24. BCA蛋白测定试剂盒(Thermo Fisher Scientific,目录号:23225)
  25. 硅化低结合末端200-1,000μl(Denville Scientific Inc.,目录号:P3193-S)
  26. 硅化低结合尖端200μl(Denville Scientific Inc.,目录号:P3010-S)
  27. 蛋白A/G PLUS-琼脂糖非缀合珠(Santa Cruz Biotechnology,目录号:SC-2003)
  28. EZview Red ANTI-FLAG M2亲和凝胶结合珠(Sigma-Aldrich,目录号:F2426)
  29. GFP多克隆抗体,兔(Genscript,目录号:A01388)
  30. GFP单克隆抗体(B-2)(小鼠)(Santa Cruz Biotechnology,目录号:SC-9996)
  31. 驴抗兔IgG-HRP(Santa Cruz Biotechnology,目录号:SC-2313)
  32. 山羊抗小鼠IgG-HRP(Santa Cruz Biotechnology,目录号:SC-2005)
  33. MYOB干混合(见配方)
  34. MYOB板(见配方)
  35. M9缓冲区(请参阅配方)
  36. 裂解缓冲液(见配方)
  37. SDS-PAGE样品缓冲液(参见配方)

设备

  1. 冷冻微量离心机(Eppendorf,型号:5417R)
  2. 大型离心机(Eppendorf,型号:5810R)
  3. 旋转式搅拌器(Barnstead Thermolyne LabQuake,型号:C415110)
  4. 超声波仪(Branson Sonifier,型号:450)
  5. SDS-PAGE样品缓冲液(参见配方)

设备

  1. 冷冻微量离心机(Eppendorf,型号:5417R)
  2. 大型离心机(Eppendorf,型号:5810R)
  3. 旋转式搅拌器(Barnstead Thermolyne LabQuake,型号:C415110)
  4. 超声波仪(Branson Sonifier,型号:450)
  5. ...
  6. Resuspend worms by gentle inversion in 15 ml PBS.
  7. Spin down worms and remove as much supernatant as possible.
  8. Transfer up to 500 μl of packed worms using siliconized tips into a microcentrifuge tube.
    Note: Siliconized tips are recommended to prevent adherence of worms.
  9. Add up to 1 ml of ice cold lysis buffer (2x worm volume) and incubate on ice for 30 min.
    Note: Worms can be flash frozen using liquid nitrogen and stored at -80 °C up to one week. For best results, continue with lysis immediately.

  • Lysis of worms
    1. If performing lysis on same day, flash freeze worms in liquid nitrogen for 10 sec. If performing lysis on previously stored worm samples, continue to step B2.
    2. Thaw frozen worms in room temperature water until a third of the frozen sample has melted.
    3. Insert tip of sonicator halfway into sample and sonicate for 5-7 sec using 8 W power. Immediately flash freeze samples for 10 sec in liquid nitrogen.
      Note: During sonication, do not allow samples to completely thaw during this process. It is important to keep the sample cold at all times to avoid protein degradation. Incubating samples on ice during sonication is optimal. One alternative that has been 以前报道(Ding等人,2005)是使用杵和研钵   在液氮存在下粉碎蠕虫小球以破碎   角质层,这可能有助于增加蛋白质产量和保存 蛋白质的完整性。
    4. 重复步骤B2-3两次。 最后快速冷冻后,让样品在室温下解冻,直到半融化
    5. 通过端到端搅拌在4℃下旋转样品30分钟
    6. 在4℃下,以13,000rpm旋转样品30分钟
    7. 将上清(溶解的蛋白质)转移到新的 微量离心管并使用标准测量蛋白质浓度 试剂盒(例如 BCA蛋白测定试剂盒)。

  • 免疫沉淀
    1. 对于每个IP使用至少4mg的总蛋白。 8-10 mg蛋白质 建议。 用冰将蛋白质样品的体积调节至1ml 冷裂解缓冲液 注意:建议不要在此步骤中存储要冻结的样品。
    2. 平衡琼脂糖珠通过在裂解缓冲液中洗涤三次,使用2x体积的珠浆液
      1. 如果使用抗体偶联的珠,添加20-30微升平衡 珠到蛋白质样品,并在4℃下搅拌孵育2-4小时 注意:在4小时后孵育珠子没有改变我们手中的免疫沉淀效率。
      2. 如果使用非共轭珠,首先向样品中加入抗体 在4℃下搅拌孵育1小时。 然后加入20-30μl 平衡的珠,并进一步温育1-3小时 注意:我们 有使用两个抗体结合的琼脂糖珠和 非结合琼脂糖珠。 我们没有检测到任何显着的差异 IP效率之间。
    3. 孵育后,用洗珠5次   1ml冰冷的裂解缓冲液,允许样品在搅拌下温育   在4℃下洗涤2-5分钟
    4. 用50-100μlSDS-PAGE样品缓冲液洗脱蛋白质,煮沸5分钟,通过Western印迹分析 注意:
      1. 当进行蛋白质印迹时,推荐另外5-10分钟煮沸以从珠子洗脱蛋白质。
      2. 对于样品制备的Western印迹的几个实施例 使用这个协议,请参考Chan et al。 (2014年)。 特异性   的免疫沉淀将取决于特异性的程度 的抗体。 为了获得最好的结果,预清除裂解液   一种不相关的抗体以提高信噪比。 而不管, 可以执行几种不同类型的控制来评估 特异性的免疫沉淀,包括进行 用不含有表位的样品进行免疫沉淀 兴趣(Chan等,2014),或包括模拟免疫沉淀 对照,其中抗体不添加到样品前 免疫沉淀。
  • 食谱

    注意:MYOB和M9的食谱取自Burns et al。 (2006)。

    1. MYOB干混物(对于370g)
      27.5g盐酸 12克三价碱基
      230克细菌用胰蛋白胨 100克NaCl
      0.4克胆固醇(95%)
    2. MYOB版(1升)
      7.4g MYOB干混物
      24克琼脂 最多加1 L ddH 2 2 O
      高压灭菌并倒入35毫升/10厘米板
    3. M9缓冲液(1升)
      6g Na 2 HPO 4
      3g KH sub 2 PO 4 sub
      5克NaCl
      最多加1 L ddH 2 2 O
      冷却至室温后,加入2ml 1M MgSO 4 7H 2 O 2 /
    4. 裂解缓冲液
      25mM Tris-HCl(pH7.5) 100 mM NaCl
      1mM EDTA
      0.5%NP-40
      1mM PMSF
      1mM Na 3 VO 4 sub。
      1μg/ml胃酶抑素-A
      10mM NaF 1片/50ml缓冲液cOmplete蛋白酶抑制剂混合物片剂
      ddH 2到所需最终量
    5. SDS-PAGE样品缓冲液
      100 mM Tris-HCl(pH 6.8)
      2%SDS
      20%甘油 4%β-巯基乙醇 0.02%溴酚蓝

    致谢

    这项工作得到加拿大癌症学会研究所(#020511)和加拿大健康研究所(#258898)授予PJR的支持。

    参考文献

    1. Burns,A.R.,Kwok,T.C.,Howard,A.,Houston,E.,Johanson,K.,Chan,A.,Cutler,S.R.,McCourt,P.and Roy,P.J。(2006)。 在Caenorhabditis elegans中用于生物活性和靶标识别的小分子的高通量筛选。 Nat Protoc 1(4):1906-1914。
    2. Chan,K.K.,Seetharaman,A.,Bagg,R.,Selman,G.,Zhang,Y.,Kim,J.and Roy,P.J。(2014)。 EVA-1充当UNC-40共受体,以增强对MADD-4指导的吸引力 PLoS Genet 10(8):e1004521。
    3. Ding,L.,Spencer,A.,Morita,K.and Han,M。(2005)。 发育时间调节因子AIN-1与miRISCs相互作用,可能靶向argonaute 蛋白ALG-1到C中的细胞质P体。 elegans 。 Mol Cell 19(4):437-447。
    4. Hall,R.A。(2005)。 共免疫沉淀作为评价受体 - 受体或受体 - 蛋白相互作用的策略。 在:George,S.R。,和Dowd,B.F。(eds)。 < em>蛋白质偶联受体 - 蛋白质相互作用。 John Wiley& Sons,Inc.,165-178。
    • English
    • 中文翻译
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    Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
    引用:Chan, K. K., Seetharaman, A., Selman, G. and Roy, P. J. (2015). Immunoprecipitation of Proteins in Caenorhabditis elegans. Bio-protocol 5(7): e1436. DOI: 10.21769/BioProtoc.1436.
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    Mohammed Lakdawala
    Texas Woman's University
    I am trying to extract protein from C. elegans. I am using 15 10 cm confluent plates and sonicating it according to the protocol. But the protein yield is very low ~0.2 mg/mL. I have also tried using the alternative method from Ding et al, 2005, but the protein yield is the same. Can you help me figure out why is that so? I have tried using ~28 confluent 10 cm plates but there is not much increase in protein yield (~0.25mg/mL). Any help will be appreciated.

    Thanks,
    Farhan
    5/25/2017 2:04:27 PM Reply