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

Ana  Lechuga; Mónica  Berjón-Otero; Margarita  Salas; Modesto  Redrejo-Rodríguez

doi:10.21769/BioProtoc.2678

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Peer-reviewed

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

AL Ana Lechuga

MB Mónica Berjón-Otero

MS Margarita Salas email

Modesto Redrejo-Rodríguez email

发布: 2018年01月05日第8卷第1期 DOI: 10.21769/BioProtoc.2678 浏览次数: 11274

评审: Dennis NürnbergSeda EkiciAnonymous reviewer(s)

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Cover of Journal of Virology, featuring study using the protocol.

Jan 2013

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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

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. cOmplete^TM 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)
Co-immunoprecipitation
1. 1.5 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.690 )
2. Pierce^® Anti-HA agarose (Thermo Fisher Scientific, Thermo Scientific^TM, 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)
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. SeeBlue^TM Plus2 Pre-stained Protein Standard (Thermo Fischer Scientific, Invitrogen^TM, 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. ECL^TM 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

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

Procedure

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如何引用

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.