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Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay
蛋白质pull-down免疫共沉淀实验分析病毒DNA结合蛋白质之间的直接相互作用   

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本实验方案简略版
Journal of Virology
Jan 2013

Abstract

This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules out nucleic acid-mediated indirect interaction. Then, a second immunoprecipitation step is performed using the purified putative partner protein. Co-immunoprecipitated proteins can be detected either by Coomassie Blue staining and/or Western blotting (WB) if a specific antibody is available. Moreover, many DNA/RNA binding proteins are highly electropositive, which can hinder WB under standard conditions, as has been shown in histones and histone-like proteins. In this case, we show that the high isoelectric point of the putative partner results in a poor transfer. Tips to troubleshot WB transfer of highly electropositive DNA-binding proteins are provided.

Keywords: Co-immunoprecipitation (免疫共沉淀), Pull-down (Pull-down), Protein-protein interaction (蛋白质-蛋白质相互作用), Western-blot (免疫印迹), DNA-binding protein (DNA结合蛋白)

Background

Co-immunoprecipitation is a commonly used method to analyze protein-protein interactions (PPIs). Many co-immunoprecipitation protocols use bacteria-expressed proteins. However, the use of cell extracts does not preclude indirect interactions mediated by third proteins or, in the case of DNA/RNA binding proteins, nucleic acids.

Terminal protein of tectivirus Bam35 (B35TP) contains the conserved Tyrosine 194 that provides the OH group to anchor the first 5’-dTMP of the viral genome during protein-primed DNA replication. Moreover, B35TP has strong DNA-binding capacity and, like many DNA-binding proteins, it has a very high isoelectric point (about 10.6), which affect its stability and function in vitro (Berjón-Otero et al., 2016).

The aim of this protocol was to confirm the direct interaction between B35TP and the viral protein P1, a putative transcription factor, originally detected in a genome-wide yeast two hybrid screening (Berjón-Otero et al., 2017). We avoided indirect interactions mediated by nucleic acids by using benzonase-treated cell extracts to immunoprecipitate the bait protein (P1) and the purified recombinant protein as the interacting partner (B35TP).

Materials and Reagents

  1. Bacterial cell-free extract preparation
    1. 1.5 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.690 )
    2. E. coli BL21(DE3) (New England Biolabs, catalog number: C2527I )
    3. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    4. LB Broth (Sigma-Aldrich, catalog number: L3522 )
    5. Ampicillin (Sigma-Aldrich, catalog number: A9518 )
    6. Glucose (Sigma-Aldrich, catalog number: G5516 )
    7. TYM-5052 autoinduction medium (Studier, 2005) (ForMedium, catalog number: AIMLB0205 )
    8. cOmpleteTM ULTRA Tablets, EDTA-free, glass vials Protease Inhibitor Cocktail (Sigma-Aldrich, Roche Diagnostics, catalog number: 05892953001 )
    9. Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L6876 )
    10. Benzonase® Nuclease (Sigma-Aldrich, catalog number: E1014 )
    11. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
    12. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
    13. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
    14. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
    15. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
    16. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: 1551139 )
    17. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 435570 )
      Note: This product has been discontinued.
    18. Phosphate buffered saline pH 8 (PBS pH 8) (see Recipes)

  2. Co-immunoprecipitation
    1. 1.5 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.690 )
    2. Pierce® Anti-HA agarose (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26181 )
    3. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A4503 )
    4. Glycine (Sigma-Aldrich, catalog number: G8898 )
    5. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
    6. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
    7. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
    8. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: 1551139 )
    9. Tween® 20 (Sigma-Aldrich, catalog number: P1379 )
    10. Sodium dodecyl sulfate (SDS) (AppliChem, catalog number: A2572 )
    11. Trizma base (Sigma-Aldrich, catalog number: T1503 )
    12. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
    13. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    14. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
    15. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 435570 )
    16. Phosphate buffered saline pH 8 (PBS pH 8) (see Recipes)
    17. Phosphate buffered saline pH 8 with Tween (PBS-T pH 8) (see Recipes)
    18. 4x Laemmli SDS-PAGE sample buffer (see Recipes)

  3. Western-blot
    1. Immobilon-P Membrane, PVDF, 0.45 µm, 26.5 x 3.75 m roll (Merck, catalog number: IPVH00010 )
    2. Grade 3MM Chr Blotting Paper, sheet, 46 x 57 cm (GE Healthcare, catalog number: 3030-917 )
    3. X-ray film (VWR, catalog number: 11299-022 )
      Manufacturer: Associated Metals, catalog number: UPM0810 .
    4. SeeBlueTM Plus2 Pre-stained Protein Standard (Thermo Fischer Scientific, InvitrogenTM, catalog number: LC5925 )
    5. Coomassie Blue R250 (Sigma-Aldrich, catalog number: 27816 )
    6. Methanol (Sigma-Aldrich, catalog number: 32213 )
    7. Antibodies (e.g., anti-TP serum raised in rabbits, goat anti-rabbit horseradish peroxidase-conjugate antibody [GE Healthcare, catalog number: RPN4301 ])
    8. ECLTM Blotting Reagents (GE Healthcare, catalog number: RPN2109 )
    9. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
    10. Glycine for molecular biology (AppliChem, catalog number: A1067 )
    11. Sodium dodecyl sulfate (SDS) (AppliChem, catalog number: A2572 )
    12. Low fat milk powder (e.g., Nestle Sveltesse)
    13. Acrylamide/Bis Solution, 37.5:1 (40% w/v), 2.6% C (SERVA Electrophoresis, catalog number: 10681.01 )
    14. Ammonium peroxodisulfate for analysis EMSURE® ACS, Reag. Ph Eur. (APS) (Merck, catalog number: 1012010500 )
    15. N,N,N’,N’-Tetramethyl ethylenediamine (TEMED) GR for analysis (Merck Millipore, catalog number: 1107320100 )
    16. Phosphate buffered saline pH 7.5 (PBS) (see Recipes)
    17. Phosphate buffered saline pH 7.5 with Tween (PBS-T) (see Recipes)
    18. 4x Laemmli SDS-PAGE sample buffer (see Recipes)
    19. Western electrophoresis buffer (see Recipes)
    20. Blocking solution (see Recipes)
    21. SDS-PAGE running buffer (see Recipes)
    22. Polyacrylamide gel with 5% stacking gel and 15% running gel (see Recipes)
    23. Transfer buffers (see Recipes)
      1. Western transfer buffer
      2. Standard transfer buffer
      3. Modified transfer buffer

Equipment

  1. 50 ml flasks (v.g. VWR, catalog number: 214-1130 )
  2. Micropipettes (Gilson, model: PIPETMAN, catalog numbers: F144801 , F144600 , F144601 and F144802 )
  3. Incubator shaker for bacterial cultures
  4. Refrigerated centrifuge (Hettich, model: MIKRO 22R )
  5. Thermomixer compact (Eppendorf, catalog number 5386000010 )
  6. Gel electrophoresis chamber (Mini-Protean®, Bio-Rad Laboratories, catalog number: 1658004 )
  7. Tank blot device (Mini Trans-Blot® Electrophoretic Transfer cell, Bio-Rad Laboratories, catalog number: 1703930 )
  8. Rotating wheel
  9. Developer (Kodak, model: X-OMAT 2000 processor ) or WB documentation system
  10. Autoradiography Cassette (Amersham Hypercassette)
  11. Sonicator (Sartorius, model: LABSONIC® M )

Procedure

This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation (Figure 1). In this protocol one of the proteins, P1 from bacteriophage Bam35, is overexpressed in E. coli as an HA-tagged recombinant protein and bound to the HA-affinity resin. Three different IPs are performed, using Nt- or Ct-tagged P1 with the HA motif (HA-P1 and P1-HA, respectively), as well as the empty plasmid as a negative control.
After a washing step, the putative partner protein, B35TP, is incubated with the P1 bound to the resin. This might also be done using both extracts or both purified proteins. We advise not to use cell extracts in the second step in order to avoid false positives derived from interactions mediated by third proteins with the HA-tagged recombinant protein.


Figure 1. Flow chart of the co-immunoprecipitation procedure

  1. Bacterial cell-free extract preparation (Figure 1A)
    1. Inoculate a starter culture of E. coli BL21(DE3) harboring the corresponding expression vector or the empty plasmid (as a control to reduce false positives) from a -80 °C glycerol stock in 10 ml LB with 150 µg/ml of ampicillin and 40 mM glucose in 50 ml flasks. Grow overnight at 37 °C, 200 rpm. Different expression vectors may require different antibiotics.
    2. Dilute saturated cultures 1:100 in 10 ml of fresh TYM-5052 media supplemented with 150 µg/ml of ampicillin and incubate at 30 °C for 16 h in 50 ml flasks. This step may require optimization for each particular recombinant protein.
    3. Harvest the bacterial cells by centrifugation for 1 min at 20,000 x g at 4 °C. If required pelleted cells could be stored at -80 °C.
    4. Resuspend pellet in 450 µl of sterile PBS pH 8 (see Recipes) supplemented with protease inhibitor.
      Notes:
      1. PBS at pH 8 was used because of the high isoelectric point of many DNA-binding proteins, which in the case of B35TP strongly affects its stability and function in vitro (Berjón-Otero et al., 2016). This may need to be optimized for each protein.
      2. Protease inhibitor tablets can be added directly to the buffer according to the manufacturer’s instructions. However, since we prefer to prepare a small volume of each buffer, we prepared a 5x stock in PBS buffer (kept at -80 °C) and diluted up to 1x prior to use.
    5. Add 50 µl of lysozyme 10 mg/ml and incubate for 20 min on ice.
    6. Disrupt the cells by sonication.
      1. Set the sonicator at amplitude of 18 µ.
      2. Sonicate the bacterial suspension on ice for 2-5 sec.
      3. Keep on ice for 15-30 sec to avoid overheating of the sample.
      4. Repeat 6-8 times Steps A6b and A6c.
    7. Add 1.5 µl of benzonase and 2.5 µl of 500 mM MgCl2 and incubate for 30 min at room temperature. Stop the reaction by adding 1.5 µl of 500 mM EDTA.
      Note: This step is essential to rule out DNA-mediated indirect interactions between DNA-binding proteins.
    8. Centrifuge for 10 min at 20,000 x g at 4 °C to pellet cellular debris.
    9. Transfer the supernatant containing the cell-free protein extract to a new 1.5 ml microcentrifuge tube. We recommend performing the immunoprecipitation on the same day.

  2. Co-immunoprecipitation
    1. Immunoprecipitation of the bait protein expressed in bacteria
      Prepare the required tubes by adding 50 µl of Anti-HA agarose to a 1.5 ml microcentrifuge tube. In this case, we used three tubes for the extracts of bacteria expressing HA-P1 and P1-HA, and the negative control.
      1. Centrifuge for 10-20 sec at 12,000 x g at 4 °C and discard the supernatant.
      2. Wash the resin twice with one resin volume of PBS pH 8 supplemented with complete protease inhibitor.
      3. Centrifuge for 10-20 sec at 12,000 x g at 4 °C and discard the supernatant.
      4. Add the bacterial cell-free extract from Procedure A to the resin. Incubate the mixture at 4 °C for 18 h on a rotating wheel.
      5. Spin briefly (10-20 sec, 12,000 x g) at 4 °C and keep the supernatant for analysis of binding efficiency. Samples for analysis can be stored either on ice or at -20 °C for a longer period of time.
      6. Wash the resin with ten volumes of PBS-T pH 8 (see Recipes) for 5 min at 4 °C on a rotating wheel. Spin briefly (10-20 sec, 12,000 x g) at 4 °C and repeat three times. Keep the supernatant of the three washing steps for analysis.
    2. Pull-down co-immunoprecipitation of putative interacting proteins
      1. Add a mixture of 2 µg of the purified putative partner protein and 30 µg of bovine serum albumin in 500 µl of PBS-T to the resin and incubate at 4 °C for 2 h on a rotating Wheel. We used purified B35TP as partner protein, which was purified and quantified as described in Berjón-Otero et al., 2016.
      2. Wash the resin as before (Steps B1f-B1g).
        Note: It is recommended to transfer the resin to a new tube before the last wash step to discard proteins that may be retained on the plastic.
      3. Elute the bound proteins with 50 µl of 1x Laemmli SDS-PAGE sample buffer (see Recipes).
        Note: According to the manufacturer’s instructions, immunoprecipitated proteins can usually be eluted with 0.1 M glycine pH 2.0-2.8 for downstream procedures.

  3. Western blotting
    1. Add 4 µl of 4x Laemmli SDS-PAGE sample buffer to 10 µl of binding and washing samples.
    2. Heat all samples (elution samples included) to 95-100 °C for 3 min and spin briefly (10 sec at 10,000 x g).
    3. Load samples onto an SDS-PAGE gel (5 µl of the elution sample and the entire volume of binding and washing samples) and load 6 μl eBlueTM Plus2 Pre-stained Protein Standard to determine molecular weights.
    4. Electrophoresis at constant 150 V for one hour.
      Note: Gels can be stained by incubation for 1 h in Coomassie Blue solution and destained by incubation for 1 h in destaining solution instead of/besides doing Western blotting. For staining protocol see He (2011).
    5. Transfer to PVDF membrane using transfer buffer (see Recipes) and pieces of Grade 3MM Chr Blotting Paper (constant 100 V for 90 min at 4 °C).
      Notes:
      1. In this case, because of the high isoelectric point of the protein, a modified transfer buffer with 0.025% SDS and without methanol was used (see Recipes and Figure 2).
      2. To improve the transfer result, it is highly recommended to equilibrate both gel and membrane in the corresponding transfer buffer (5 min at room temperature).


        Figure 2. Effect of WB transfer buffer on B35TP detection. Increasing amounts of B35TP were separated by 15% SDS-PAGE and wet-transferred for 40 min either in standard transfer buffer (see Recipes) or in modified transfer buffer (see Recipes). The range of detectable protein amount may need to be optimized. WB was carried out as described in the text and, after detection (lower panels), PVDF membranes were stained with Coomassie Blue solution (upper panels).

    6. Wash membrane with PBS for 20 sec.
    7. Incubate membrane in 100% methanol for 20 sec and dry at room temperature.
    8. Incubate membrane in 20 ml of blocking solution (see Recipes) for 30 min at room temperature or overnight at 4 °C.
    9. Incubate membrane with the primary antibody (concentration depends on the antibody) in 10 ml antibody dilution buffer with gentle agitation for 1 h at room temperature.
    10. Wash three times for 10 min each with 20 ml of PBS-T.
    11. Incubate membrane with the secondary antibody (concentration depends on the antibody) in 10 ml antibody dilution buffer with gentle agitation for 1 h at room temperature.
    12. Wash five times for 5 min each with 20 ml of PBS-T.
    13. Incubate membrane with 2 ml ECLTM Blotting Reagents for 1 min at RT.
    14. Drain membrane of excess developing solution, wrap in plastic wrap and expose to X-ray film.

Data analysis

Samples from each step should be analyzed by either SDS-PAGE followed by Coomassie staining and/or WB.

Recipes

Note: Except otherwise indicated, all the solutions can be stored at room temperature for a few months.

  1. Phosphate buffered saline, pH 7.5/8 (PBS pH 7.5/8)

    Mix in 800 ml dH2O, adjust pH to 7.5 (or pH 8) with HCl, then adjust volume to 1 L
  2. Phosphate buffered saline pH 7.5/8 with Tween (PBS-T pH 7.5/8)

    Mix in 800 ml dH2O, adjust pH to 7.5 (or pH 8) with HCl, then adjust volume to 1 L
  3. 4x Laemmli SDS-PAGE sample buffer

    Mix in 4 ml dH2O, then adjust volume to 5 ml
  4. Western blot electrophoresis buffer

  5. Blocking solution (use fresh-made)
    1 g low fat milk powder
    Mix in 80 ml PBS-T, adjust volume to 100 ml
  6. SDS-PAGE running buffer 

    Mix in 800 ml dH2O, adjust volume to 1 L
  7. Polyacrylamide gel with 5% stacking gel and 15% running gel

  8. Transfer buffers (amount for 1 L)

    Mix in 800 ml dH2O, adjust volume to 1 L

Acknowledgments

This protocol describes the methodology used in the original paper (Berjón-Otero et al., 2017). This work was supported by Spanish Ministry of Economy and Competitiveness [BFU2014-52656P to M.S.] and ComFuturo Grant from Fundación General CSIC [NewPols4Biotech to M.R-R.]. M.B-O. and A.L. were holders of PhD fellowships FPI [BES-2012-052228] and FPU [15/05797] from the Spanish Economy and Competitiveness and Education Ministries, respectively. An institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa is also acknowledged. The authors do not have any conflict of interest or competing interests to declare

References

  1. Berjón-Otero, M., Lechuga, A., Mehla, J., Uetz, P., Salas, M. and Redrejo-Rodriguez, M. (2017). Bam35 tectivirus intraviral interaction map unveils new function and localization of phage ORFan proteins. J Virol.
  2. Berjón-Otero, M., Villar, L., Salas, M. and Redrejo-Rodriguez, M. (2016). Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35. Nucleic Acids Res 44(20): 9733-9744.
  3. He, F. (2011). Coomassie blue staining. Bio Protoc e78.
  4. Studier, F. W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1): 207-234.

简介

该协议通过下拉共免疫沉淀分析两种DNA结合蛋白之间的直接相互作用。其中一种蛋白在E中过表达。如HA标记的重组蛋白和无细胞提取物在HA亲和树脂中免疫沉淀。用核酸酶处理细胞提取物以降解DNA和RNA,这排除了核酸介导的间接相互作用。然后,使用纯化的推定的配偶体蛋白进行第二次免疫沉淀步骤。如果特异性抗体可用,可以通过考马斯蓝染色和/或Western印迹(WB)检测免疫共沉淀蛋白质。此外,许多DNA / RNA结合蛋白具有高度正电性,在标准条件下可阻碍WB,正如组蛋白和组蛋白样蛋白所示。在这种情况下,我们表明,假定的合作伙伴的高等电点导致转移不良。提示麻烦WB提供高正电荷DNA结合蛋白的转移。


【背景】共免疫沉淀是分析蛋白质 - 蛋白质相互作用(PPI)的常用方法。许多共免疫沉淀方案使用细菌表达的蛋白质。然而,细胞提取物的使用不排除由第三种蛋白介导的间接相互作用,或者在DNA / RNA结合蛋白的情况下介导核酸。

乙型病毒Bam35(B35TP)的末端蛋白含有保守的酪氨酸194,其提供OH基团以在蛋白质引发的DNA复制期间锚定病毒基因组的第一个5'-dTMP。此外,B35TP具有很强的DNA结合能力,与许多DNA结合蛋白一样,它具有非常高的等电点(约10.6),这影响其体外稳定性和功能(Berjón-Otero 等),2016)。

该方案的目的是确认B35TP与最初在全基因组酵母双杂交筛选中检测到的推定转录因子病毒蛋白P1(Berjón-Otero等人, 2017年)。我们通过使用benzonase处理的细胞提取物免疫沉淀诱饵蛋白(P1)和纯化的重组蛋白作为相互作用配偶体(B35TP)来避免由核酸介导的间接相互作用。

关键字:免疫共沉淀, Pull-down, 蛋白质-蛋白质相互作用, 免疫印迹, DNA结合蛋白

材料和试剂

  1. 细菌无细胞提取物制备
    1. 1.5 ml微量离心管(SARSTEDT,目录号:72.690)
    2. 电子。 (New England Biolabs,目录号:C2527I)
    3. 甘油(Sigma-Aldrich,目录号:G5516)
    4. LB肉汤(Sigma-Aldrich,目录号:L3522)
    5. 氨苄青霉素(Sigma-Aldrich,目录号:A9518)
    6. 葡萄糖(Sigma-Aldrich,目录号:G5516)
    7. TYM-5052自动感应介质(Studier,2005)(ForMedium,目录号:AIMLB0205)
    8. 无EDTA的玻璃瓶蛋白酶抑制剂混合物(Sigma-Aldrich,Roche Diagnostics,目录号:05892953001)
    9. 来自鸡蛋白的溶菌酶(Sigma-Aldrich,目录号:L6876)
    10. Benzonase核酸酶(Sigma-Aldrich,目录号:E1014)
    11. 氯化镁六水合物(MgCl 2•6H 2 O)(Sigma-Aldrich,目录号:M2670)
    12. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
    13. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
    14. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
    15. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S7907)
    16. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:1551139)
    17. 盐酸(HCl)(Sigma-Aldrich,目录号:435570)
      注:此产品已停产。
    18. 磷酸盐缓冲盐水pH 8(PBS pH 8)(见食谱)

  2. 免疫共沉淀
    1. 1.5 ml微量离心管(SARSTEDT,目录号:72.690)
    2. Pierce Anti-HA琼脂糖(Thermo Fisher Scientific,Thermo Scientific TM,目录号:26181)
    3. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A4503)
    4. 甘氨酸(Sigma-Aldrich,目录号:G8898)
    5. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
    6. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
    7. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S7907)
    8. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:1551139)
    9. Tween 20(Sigma-Aldrich,目录号:P1379)
    10. 十二烷基硫酸钠(SDS)(AppliChem,目录号:A2572)
    11. Trizma碱(Sigma-Aldrich,目录号:T1503)
    12. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
    13. 甘油(Sigma-Aldrich,目录号:G5516)
    14. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
    15. 盐酸(HCl)(Sigma-Aldrich,目录号:435570)
    16. 磷酸盐缓冲盐水pH 8(PBS pH 8)(见食谱)
    17. 用吐温(PBS-T pH 8)的磷酸盐缓冲盐水pH 8(见食谱)
    18. 4x Laemmli SDS-PAGE样品缓冲液(见食谱)

  3. 免疫印迹
    1. Immobilon-P膜,PVDF,0.45μm,26.5×3.75m卷(Merck,目录号:IPVH00010)
    2. 等级3MM Chr印刷纸张,46 x 57厘米(GE Healthcare,产品目录号:3030-917)
    3. X光胶片(VWR,目录号:11299-022)
      制造商:Associated Metals,产品目录号:UPM0810。
    4. SeeBlue TM Plus2预染色蛋白标准品(Thermo Fischer Scientific,Invitrogen TM,目录号:LC5925)
    5. 考马斯蓝R250(Sigma-Aldrich,目录号:27816)
    6. 甲醇(Sigma-Aldrich,目录号:32213)
    7. 在兔子中产生的抗TP血清,山羊抗兔辣根过氧化物酶结合物抗体[GE Healthcare,产品目录号:RPN4301])。
    8. ECL TM印迹试剂(GE Healthcare,目录号:RPN2109)
    9. Trizma base(Sigma-Aldrich,目录号:T1503)
    10. 用于分子生物学的甘氨酸(AppliChem,目录号:A1067)
    11. 十二烷基硫酸钠(SDS)(AppliChem,目录号:A2572)
    12. 低脂奶粉(如,雀巢斯维特斯)
    13. 丙烯酰胺/ Bis溶液,37.5:1(40%w / v),2.6%C(SERVA电泳,目录号:10681.01)
    14. 用于分析的过氧二硫酸铵EMSURE ACS,Reag。 Ph Eur。 (APS)(Merck,目录号:1012010500)
    15. N,N,N',N' - 四甲基乙二胺(TEMED)GR用于分析(Merck Millipore,目录号:1107320100)
    16. 磷酸盐缓冲盐水pH 7.5(PBS)(见食谱)
    17. 用吐温(PBS-T)的磷酸盐缓冲盐水pH7.5(见食谱)
    18. 4x Laemmli SDS-PAGE样品缓冲液(见食谱)
    19. 西方电泳缓冲液(见食谱)
    20. 阻止解决方案(见食谱)
    21. SDS-PAGE运行缓冲液(见食谱)
    22. 聚丙烯酰胺凝胶5%浓缩胶和15%运行凝胶(见食谱)
    23. 传输缓冲区(请参阅食谱)
      1. 西方传输缓冲区
      2. 标准传输缓冲区
      3. 修改传输缓冲区

设备

  1. 50毫升烧瓶(例如VWR,目录号:214-1130)
  2. 微量移液器(Gilson,型号:PIPETMAN,产品目录号:F144801,F144600,F144601和F144802)
  3. 培养箱用于细菌培养
  4. 冷冻离心机(海蒂诗,型号:MIKRO 22R)
  5. 紧凑型Thermomixer(Eppendorf,目录号5386000010)
  6. 凝胶电泳室(Mini-Protean,Bio-Rad Laboratories,目录号:1658004)
  7. 油墨印迹装置(Mini Trans-Blot电泳转移池,Bio-Rad Laboratories,目录号:1703930)
  8. 旋转轮
  9. 开发者(柯达,型号:X-OMAT 2000处理器)或WB文档系统
  10. 放射自显影盒(Amersham Hypercassette)
  11. Sonicator(Sartorius,型号:LABSONIC M)

程序

该方案通过下拉免疫共沉淀分析两种DNA结合蛋白之间的直接相互作用(图1)。在这个协议中,来自噬菌体Bam35的P1蛋白之一在E中过表达。将其作为HA标记的重组蛋白并与HA亲和树脂结合。使用具有HA基序的Nt-或Ct-标记的P1(分别为HA-P1和P1-HA)以及空质粒作为阴性对照进行三种不同的IP。
在洗涤步骤之后,推定的配偶体蛋白B35TP与结合到树脂上的P1一起温育。这也可以使用两种提取物或两种纯化的蛋白来完成。我们建议不要在第二步中使用细胞提取物,以避免第三种蛋白与HA标记的重组蛋白介导的相互作用导致的假阳性。


图1.共免疫沉淀过程的流程图

  1. 细菌无细胞提取物制备(图1A)
    1. 接种 E的初学者文化。从具有150μg/ ml氨苄青霉素和40mM葡萄糖的10ml LB中的-80℃甘油储液中获得含有相应表达载体或空质粒(作为减少假阳性的对照)的大肠杆菌BL21(DE3)在50毫升烧瓶中。在37℃,200rpm下过夜生长。不同的表达载体可能需要不同的抗生素。
    2. 在10毫升添加有150微克/毫升氨苄青霉素的新鲜TYM-5052培养基中稀释饱和的培养物1:100,并在30毫升的50毫升烧瓶中孵育16小时。这个步骤可能需要优化每个特定的重组蛋白。
    3. 通过在4℃下20,000xg离心1分钟来收获细菌细胞。如果需要,粒状细胞可以储存在-80°C。
    4. 重悬沉淀在450μL的无菌PBS pH值8(见食谱)补充蛋白酶抑制剂。
      注意:
      1. 由于许多DNA结合蛋白的等电点很高,所以使用pH值为8的PBS,在B35TP的情况下,其强烈影响其体外的稳定性和功能(Berjón-Otero等,2016)。这可能需要针对每种蛋白质进行优化。
      2. 蛋白酶抑制剂片剂可根据制造商的说明书直接添加到缓冲液中。然而,由于我们倾向于准备少量的每种缓冲液,所以我们在PBS缓冲液(保持在-80℃)下制备了5×储备液,并在使用前稀释至1倍。
    5. 加入50微升溶菌酶10毫克/毫升,在冰上孵育20分钟。
    6. 通过超声破碎细胞。
      1. 设置超声波振幅为18μ。
      2. 超声波在冰上的细菌悬液2-5秒。
      3. 保持在冰上15-30秒,以避免过热的样本。

      4. 重复6-8次步骤A6b和A6c
    7. 加入1.5μl的benzonase和2.5μl的500mM MgCl 2并在室温下孵育30分钟。加入1.5μl500 mM EDTA终止反应。
      注意:这一步对于排除DNA结合蛋白之间的DNA介导的间接相互作用至关重要。
    8. 20000×g离心10分钟,4°C沉淀细胞碎片。
    9. 将含有无细胞蛋白提取物的上清液转移到新的1.5ml微量离心管中。我们建议在同一天进行免疫沉淀。

  2. 免疫共沉淀
    1. 在细菌中表达的诱饵蛋白的免疫沉淀
      准备所需的管,加入50微升的抗HA琼脂糖1.5毫升微量离心管。在这种情况下,我们使用三个管来表达HA-P1和P1-HA的细菌提取物和阴性对照。
      1. 在4℃以12,000×gg离心10-20秒,弃去上清液。
      2. 用一种树脂体积的pH8的PBS清洗树脂两次,并补充完整的蛋白酶抑制剂。
      3. 在4℃以12,000×gg离心10-20秒,弃去上清液。
      4. 将来自程序A的无菌细胞提取物添加到树脂中。
        在4°C下将混合物在转轮上孵育18小时
      5. 在4℃短暂旋转(10-20秒,12,000×g克)并保持上清液用于结合效率的分析。用于分析的样品可以储存在冰上或-20°C较长时间。
      6. 用旋转的转轮在4℃下用10倍体积的PBS-T pH8(见配方)清洗树脂5分钟。在4℃短暂旋转(10-20秒,12,000×g克)并重复三次。保留三个清洗步骤的上清液进行分析。
    2. 推定共免疫沉淀推定相互作用的蛋白质
      1. 在500μlPBS-T中加入2μg纯化的推定的配偶体蛋白质和30μg牛血清白蛋白的混合物到树脂中,并在旋转的轮上在4℃孵育2小时。我们使用纯化的B35TP作为合作伙伴蛋白,按照Berjón-Otero等人2016年的描述进行纯化和定量。
      2. 像以前一样清洗树脂(步骤B1f-B1g)。
        注意:建议在最后一次清洗步骤之前将树脂转移到新的管中,以丢弃可能保留在塑料上的蛋白质。
      3. 用50μl1x Laemmli SDS-PAGE样品缓冲液洗脱结合的蛋白质(见食谱)。
        注意:根据生产商的说明,免疫沉淀蛋白通常可以用0.1M甘氨酸pH 2.0-2.8的洗脱液进行下游操作。

  3. Western blotting
    1. 加入4微升4×Laemmli SDS-PAGE样品缓冲液到10微升的结合和洗涤样品。
    2. 将所有样品(包括洗脱样品)加热至95-100℃3分钟,并短暂旋转(10万秒×10000)。
    3. 将样品加载到SDS-PAGE凝胶(5μl洗脱样品和全部结合和洗涤样品)上并加载6μleBlue TM Plus2预染色的蛋白质标准物以测定分子量。 br />

    4. 恒定电压150伏电泳1小时 注意:凝胶可以在考马斯亮蓝溶液中孵育1小时,在脱色溶液中孵育1小时而不是/除了蛋白质印迹之外,染色。对于染色方案,见He(2011)。
    5. 使用转移缓冲液(参见配方)和3MM Chr等级印刷纸(在4°C恒定100V 90分钟)转移到PVDF膜上。
      注意:
      1. 在这种情况下,由于蛋白质的等电点很高,使用了含有0.025%SDS和不含甲醇的修饰转移缓冲液(参见食谱和图2)。
      2. 为了改善转印结果,强烈建议在相应的转印缓冲液(室温下5分钟)中平衡凝胶和膜。


        图2.WB转移缓冲液对B35TP检测的影响通过15%SDS-PAGE分离增加量的B35TP,并且在标准转移缓冲液(参见配方)中或在修饰中湿转移40分钟转移缓冲区(参见食谱)。可检测的蛋白质量的范围可能需要优化。 WB如文中所述进行,检测后(下图)PVDF膜用考马斯蓝溶液染色(上图)。

    6. 用PBS洗膜20秒。
    7. 将膜在100%甲醇中孵育20秒,并在室温下干燥。
    8. 在20ml封闭液中孵育膜(见食谱)在室温下30分钟或在4℃下过夜。
    9. 用10 ml抗体稀释缓冲液孵育膜的一抗(浓度取决于抗体),在室温下温和搅拌1小时。
    10. 每次用20毫升的PBS-T洗涤三次,每次10分钟。
    11. 用10 ml抗体稀释缓冲液孵育二抗(浓度取决于抗体),在室温下温和搅拌1小时。

    12. 用20毫升PBS-T洗5次,每次5分钟
    13. 用2ml ECL TM印迹试剂在RT孵育膜1分钟。
    14. 排出过量显影液的膜,包上保鲜膜并暴露在X光胶片上。

数据分析

来自每个步骤的样品应该通过SDS-PAGE,然后考马斯染色和/或WB进行分析。

食谱

注:除非另有说明,否则所有溶液均可在室温下保存数月。

  1. 磷酸盐缓冲盐水,pH 7.5 / 8(PBS pH 7.5 / 8)


    在800ml dH 2 O中混合,用HCl调节pH至7.5(或pH 8),然后调节体积至1L。
  2. 用吐温(PBS-T pH 7.5 / 8)pH7.5 / 8的磷酸盐缓冲盐水


    在800ml dH 2 O中混合,用HCl调节pH至7.5(或pH 8),然后调节体积至1L。
  3. 4倍Laemmli SDS-PAGE样品缓冲液


    在4ml dH 2 O中混合,然后调节体积到5ml
  4. Western印迹电泳缓冲液

  5. 阻塞解决方案(使用新鲜的)
    1克低脂奶粉
    在80毫升PBS-T中混合,调整体积至100毫升
  6. SDS-PAGE运行缓冲液


    在800毫升dH 2 O 0中混合,将体积调节至1升
  7. 聚丙烯酰胺凝胶含5%浓缩凝胶和15%运行凝胶


  8. 转移缓冲液(1升量)


    在800毫升dH 2 O 0中混合,将体积调节至1升

致谢

该协议描述了原始论文中使用的方法(Berjón-Otero等人,2017)。这项工作是由西班牙经济和竞争力部[BFU2014-52656P至M.S.]和ComFuturo格兰特基金会总会CSIC [NewPols4Biotech至M.R-R]支持的。 M.B-O。和A.L.分别持有西班牙经济和竞争力与教育部门的FPD奖学金(BES-2012-052228)和FPU(15/05797)。拉蒙生物资源中心分子生物学研究中心的机构资助也得到了承认。作者没有任何利益冲突或竞争利益的申报

参考

  1. Berjón-Otero,M.,Lechuga,A.,Mehla,J.,Uetz,P.,Salas,M.和Redrejo-Rodriguez,M.(2017)。 Bam35病毒内病毒相互作用图揭示了噬菌体ORFan蛋白的新功能和定位。 > J Virol 。
  2. Berjón-Otero,M.,Villar,L.,Salas,M.和Redrejo-Rodriguez,M.(2016)。 披露革兰氏阳性病毒Bam35蛋白质引发的基因组复制的早期步骤
    核酸研究44(20):9733-9744
  3. 他,楼(2011年)。 考马斯亮蓝染色 Bio Protoc e78。
  4. Studier,F. W.(2005)。 高密度摇瓶培养中的自动诱导蛋白质生产 Protein Expr Purif 41(1):207-234。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lechuga, A., Berjón-Otero, M., Salas, M. and Redrejo-Rodríguez, M. (2018). Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay. Bio-protocol 8(1): e2678. DOI: 10.21769/BioProtoc.2678.
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