Protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step immunoprecipitation approach

Summary

Co-immunoprecipitation (coIP) is an experimental technique to study protein-protein interactions (PPIs). However, single-step coIP can only be used to identify the interaction between two proteins and does not solve the interaction testing of ternary complexes. Here, we present a protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step coIP approach. We describe steps for cell culture and transfection, elution of target proteins, and two-step coIP including western blot analyses.

For complete details on the use and execution of this protocol, please refer to Li et al.¹

Graphical abstract

Highlights

• •Protocols to identify the interaction of ternary complexes
• •Details on how to efficiently elute the target protein
• •Instruction for designing a two-step co-immunoprecipitation strategy

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Co-immunoprecipitation (coIP) is an experimental technique to study protein-protein interactions (PPIs). However, single-step coIP can only be used to identify the interaction between two proteins and does not solve the interaction testing of ternary complexes. Here, we present a protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step coIP approach. We describe steps for cell culture and transfection, elution of target proteins, and two-step coIP including western blot analyses.

Before you begin

ATG5 (Autophagy-related 5, ATG5) is a key regulator of autophagy and is involved in the formation of autophagosomes together with ATG12 in the classical process of autophagy. FBXW7 (F-box and WD repeat domain containing 7, FBXW7) is a target protein recognition component of SCF (SKP1-cullin-F-box) type ubiquitin ligase. β-TrCP is a substrate recognition receptor of the SCF E3 ubiquitin ligase complex, which is responsible for recognizing specific protein substrates and initiating their ubiquitination degradation. In our article, Flag-ATG5 recruits FBXW7 to promote ubiquitination and proteasome-mediated degradation of HA-β-TrCP1, resulting in the inhibition of nuclear factor kB (NF-kB) signaling and inflammatory response.¹ The transfected gene needs to be labeled differently, (e.g., Flag-tag, HA-tag, Myc-tag, Strep-Tag Ⅱ, ALFA-tag). This protocol below describes the specific steps for two-step coIP test for the ternary complexes (Figure 1), and it can also be used for protein interactions in vitro.

Figure 1.

Two-step coIP assay working model

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	Storage conditions
Antibodies
Rabbit anti-HA (1:1,000)	CST	Cat# 3724; RRID: AB_1549585	−20°C
Rabbit anti-Flag (1:1,000)	CST	Cat# 14793; RRID: AB_2572291	−20°C
Rabbit anti-FBXW7 (1:1,000)	Abcam	Cat# ab109617; RRID: AB_2687519	−20°C
HRP-conjugated secondary antibodies (1:5,000)	ZSGB-Bio	Cat# ZB-2301; RRID: AB_2747412	−20°C
Chemicals, peptides, and recombinant proteins
Flag peptide	Sangon Biotech	Cat# T510060	−80°C
DMEM	HyClone	Cat# SH30243.01	4°C
FBS	TIANHANG	Cat# 11011-8611	−20°C
Lipo8000	Beyotime	Cat# C0533	−20°C
RIPA buffer	Beyotime	Cat# P0013D	−20°C
PMSF	Beyotime	Cat# ST506	−20°C
ECL	CWBIO	Cat# CW0049M	4°C
Experimental models: Cell lines
HEK293T cells	Chinese National Collection of Authenticated Cell Lines	Cat# GNHu17	−80°C
Recombinant DNA
Flag-ATG5 plasmid	This paper	N/A	−20°C
HA-β-TrCP1 plasmid	This paper	N/A	−20°C
Other
Anti-Flag magnetic beads	Beyotime	Cat# P2115	4°C
Anti-HA magnetic beads	Beyotime	Cat# P2121	4°C
Anti-IgG magnetic beads	Beyotime	Cat# P2171	4°C
Protein electrophoresis system	Bio-Rad	Mini-PROTEAN Tetra	N/A
Chemiluminescence gel imaging system	Bio-Rad	ChemiDoc XRS+	N/A
Rotary mixer	Thermo Fisher Scientific	Cat# 88881002	N/A

Materials and equipment

Buffers and other solutions

Elution buffer

Reagent	Final concentration	Amount
NaCl	250 mM	1.4625 g
ddH2O	N/A	Up to 100 mL
Total	N/A	100 mL

Stored at 15–25°C for 1 year.

TBST

Reagent	Final concentration	Amount
NaCl	150 mM	8.8 g
1 M Tris-HCl (pH 8.0)	20 mM	20 mL
Tween 20	0.05% (V/V)	0.5 mL
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Stored at 15–25°C for 1 year.

5 × SDS-PAGE loading buffer

Reagent	Final concentration	Amount
1 M Tris-HCl (pH 6.8)	250 mM	12.5 mL
SDS	10% (W/V)	5 g
Bromophenol blue	0.5% (W/V)	250 mg
Glycerol	50% (W/V)	25 mL
ddH2O	N/A	Up to 50 mL
Total	N/A	50 mL

Add β-Mercaptoethanol just before use. Final concentration 5% (V/V). Stored at 15–25°C for 1 month.

10 × Running Buffer

Reagent	Final concentration	Amount
SDS	1% (W/V)	10 g
Tris	250 mM	30.2 g
Glycine	2.5 M	188 g
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Stored at 15–25°C for 1 year.

10 × Transfer Buffer

Reagent	Final concentration	Amount
SDS	0.37% (W/V)	3.7 g
Tris	480 mM	58 g
Glycine	390 mM	29 g
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Add methanol just before use. Final concentration 20% (V/V). Stored at 15–25°C for 1 month.

1 M Tris-HCl (pH 6.8)

Reagent	Final concentration	Amount
Tris	1 M	121.1 g
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Add HCl to pH = 6.8. Stored at 4°C for 1 year.

1.5 M Tris-HCl (pH 8.8)

Reagent	Final concentration	Amount
Tris	1.5 M	181.7 g
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Add HCl to pH = 8.8. Stored at 4°C for 1 year.

10% AP solution

Reagent	Final concentration	Amount
Ammonium persulfate	10% (W/V)	5 g
ddH2O	N/A	Up to 50 mL
Total	N/A	50 mL

Stored at 4°C for 2 weeks.

10% SDS solution

Reagent	Final concentration	Amount
Sodium dodecyl sulfate	10% (W/V)	5 g
ddH2O	N/A	Up to 50 mL
Total	N/A	50 mL

Stored at 15–25°C for 1 year.

30% Acrylamide solution

Reagent	Final concentration	Amount
Acrylamide	29% (W/V)	290 g
Bis-Acrylamide	1% (W/V)	10 g
ddH2O	N/A	Up to 1 L
Total	N/A	1 L

Remove impurities with a 0.45 μm filter. Store at 4°C away from light.

CRITICAL: Acrylamide and bis-Acrylamide is an irritant, toxic, and carcinogenic chemical. Prepare the 30% Acrylamide solution in a fume hood with proper personal protective equipment.

Step-by-step method details

Cell culture and transfection

Timing: 3 days

Before you begin this section, you need to prepare transfection reagents and plasmids. The transfection efficiency of the plasmid will affect the quality of results. Besides, RIPA cell lysate containing PMSF needs to be prepared.

• 1.Culture the HEK293T human embryonic kidney cells at 37°C in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in a 5% CO₂ incubator.
• 2.Grow HEK293T cells to 70%–80% confluence in a 10 cm dish (other cell types may need optimization).

Note: The solutions required in the following steps are intended for a 10 cm dish.

• 3.Transfect cells with constructs (Flag-ATG5 and HA-β-TrCP1) using Lipo8000 transfection reagent according to the manufacturer’s standard protocol. The transfection volume of each group was 15 μg (https://www.beyotime.com/product/C0533-1.5ml.htm).

Note: The transfect cells with Flag and HA empty vector as the negative control.

CRITICAL: Lipo8000 transfection reagent can be replaced with Lipo2000, Lipo3000, PEI, etc.

• 4.
  Culture cells in fresh medium after transfection of 6 h.

  Note: At 48 h of transfection, the number of cells at this point is about 5×10⁶.

  • a.
    Carefully discard the medium.
  • b.
    Wash the cells with 1 mL cold PBS gently.
  • c.
    Discard the PBS completely.
• 5.Wash the cells two more times, and discard the PBS completely.
• 6.Lyse the cells with 800 μL RIPA lysis buffer containing 1 mM PMSF, and collect the cell lysates in a 1.5 mL microcentrifuge tube.
• 7.
  Place the cell lysates on ice for 30 min and sonicate three times until they are clarified.

  Note: We used a QSonica Q125 (with a 1/8 inch tip), whose maximum power is 125 W. Many other suitable sonicators can be used for this protocol.

  • a.
    Sonicate cell lysates in an ice/water slurry bath using a sonicator.
  • b.
    Sonicator setting: power is 30%. Sonicate cell lysates with 10 s on/10 min off intervals until the mixture becomes clear.
  • c.
    The above parameters can be used as the initial parameters for optimization.
• 8.Centrifuge the lysate at 4 °C at 13400 g for 10 min and collect the supernatant.

Pause point: Store the supernatant at −80°C for further analysis.

• 9.Take 100 μL of the collected supernatant (step 8) to 1.5 mL microcentrifuge tube as the input samples. And use the remaining supernatant as sample 1.
• 10.Add 25 μL 5 × SDS-PAGE loading buffer to Input samples and analyze the protein expression by western blotting (Figure 2A). 20 μg protein was added to each group.

Note: Store the input samples at −80°C for the further western blot analysis.

Figure 2.

A two-step coIP assay was performed to test for ternary complexes formation

The procedures for the two-step coIP assay are outlined on the left. HEK293T cells are transfected with Flag-tagged ATG5 (Flag-ATG5) and HA-tagged β-TrCP1 (HA-β-TrCP1). Transfected with Flag and HA empty vector cells are used as a negative control. The first coIP step is performed with anti-Flag antibodies. The complexes are eluted using the Flag peptide. In the second step of coIP, anti-HA antibodies or control IgG are used to precipitate the complexes. Protein samples from each step are then separately subjected to western blot analyses using anti-Flag, anti-HA, and anti-FBXW7 antibodies as indicated. This figure is from previously published data¹ (Figure 5L) and is reprinted with permission.

One-step coIP

Timing: 1 day

Enrichment of Flag-ATG5 protein and its protein complex by Flag magnetic beads.

• 11.Prepare the Flag magnetic beads as per steps 12–15.
• 12.Gently shake the bottle of Flag magnetic beads to resuspend the slurry.
• 13.Transfer 20 μL beads to a 1.5 mL microcentrifuge tube.
• 14.Wash the beads by adding 1 mL TBST supplemented with 1 mM PMSF and invert to mix. Using the magnetic separation rack to separate the beads and supernatant, and carefully discard the supernatant.
• 15.Wash the beads two more times (refer to step 14). Resuspend the Flag beads in 20 μL RIPA lysis buffer.
• 16.Mix the sample 1 from step 9 with the prepared beads from step 15 together in a 1.5 mL microcentrifuge tube and gently rotate for approximately 12 h at 4°C.
• 17.Using the magnetic separation rack to separate the beads and supernatant, carefully discard the supernatant, and collect the beads.
• 18.Wash the beads by adding 1 mL TBST supplemented with 1 mM PMSF and resuspend gently.
• 19.Rotate at 4°C for 10 min and collect the beads using the magnetic separation rack. Carefully discard the supernatant.
• 20.Repeat steps 18–19 two more times for a total of three washes.

Elution of the target protein

Timing: 1 day

Elution of the Flag-ATG5 protein and its protein complex bound to magnetic beads by Flag peptide.

• 21.Add 300 μL elution buffer supplemented with 100 μg/mL Flag peptide to the beads. Resuspend the beads by gently pipetting several times.
• 22.Rotate at 4°C for 3 h. Separate the beads and supernatant using the magnetic separation rack, and carefully collect the supernatant and beads.

Note: Collect the supernatant and transfer to another 1.5 mL microcentrifuge tube. The supernatant contains the target protein.

• 23.Collect the beads from step 22. Repeat steps 21 and 22, and combine the supernatants to 600 μL.
• 24.Take 100 μL supernatant from step 23 and transfer it to a new 1.5 mL microcentrifuge tube as the Flag elution sample.

Note: The remaining supernatant should not be discarded and needs to be used as sample 2 for subsequent steps.

• 25.Add 25 μL 5 × SDS-PAGE loading buffer to the Flag elution sample and analyze the protein expression using western blot (Figure 2B).

CRITICAL: The elution time of 4°C rotation should not be less than 3 h; otherwise, the elution efficiency will be affected, leading to lower production of target proteins. The elution time can be extended to improve the elution efficiency.

Two-step coIP

Timing: 2 days

Enrichment of HA-β-TrCP1 protein and its protein complex by HA magnetic beads.

• 26.Prepare the IgG beads, according to steps 12–15.
• 27.Add 20 μL IgG beads to sample 2.
• 28.Fix sample 2 on the rotation mixer and rotate at 4°C for 12 h.
• 29.Separate beads and supernatant using the magnetic separation rack, and carefully collect the supernatant and IgG beads. Use the supernatant as sample 3.
• 30.Wash the IgG beads (collected from step 29) by adding 1 mL TBST supplemented with 1 mM PMSF and resuspend gently.
• 31.Rotate at 4°C for 10 min, separate the beads and supernatant using the magnetic separation rack, and carefully discard the supernatant.
• 32.Repeat steps 30 and 31 two more times for a total of three washes.
• 33.Add 100 μL RIPA lysis buffer and resuspend IgG beads as the IP-IgG sample.

Note: IgG in the coIP system is to remove the effect of non-specific binding proteins on the results, so lgG is usually used as a negative control to further prove the credibility of the results.

• 34.Prepare the HA beads, according to steps 12–15.
• 35.Add 20 μL HA beads to sample 3.
• 36.Fix sample 3 on the rotation mixer and rotate at 4°C for 12 h.
• 37.Collect the beads using the magnetic separation rack, and carefully discard the supernatant.
• 38.Wash the beads by adding 1 mL TBST supplemented with 1 mM PMSF and resuspend gently.
• 39.Rotate at 4°C for 10 min. Carefully discard the supernatant using the magnetic separation rack.
• 40.Repeat steps 38 and 39 two more times for a total of three washes.
• 41.Add 100 μL RIPA lysis buffer and resuspend HA beads as the IP-HA sample.
• 42.Add 25 μL 5 × SDS-PAGE loading buffer to the IP-IgG sample and IP-HA sample, and analyze the protein expression using western blot (Figure 2C).

Expected outcomes

The high specificity of two-step coIP facilitates the identification of specific ternary protein complexes interactors. In this protocol, the first coIP step is performed with anti-Flag magnetic beads, the obtained complex beads are eluted with Flag peptide. In the second step of coIP, anti-HA magnetic beads and control IgG are used to precipitate the eluted complexes. Finally, protein samples from each step are then separately subjected to western blot analyses using anti-Flag, anti-HA, and anti-FBXW7 antibodies as indicated.

Limitations

This protocol can only detect the protein ternary complexes interactions, but it cannot be applied to study protein-nucleic acid interactions.

Troubleshooting

Problem 1

Low elution efficiency of target protein in step 22.

Potential solution

To ensure a quantitative release of the complexes, the elution time of 4°C rotation can be extended to more than 3 h, or reduce the volume of elution buffer to improve the elution efficiency.

Problem 2

Cells are difficult to be transfected.

Potential solution

The transfection efficiency can be improved by virus infection or create the stable expression of cell lines.

Problem 3

The efficiency of magnetic beads binding target protein is low.

Potential solution

The magnetic beads can be replaced by agarose beads combined with antibodies.

Problem 4

The concentration of target protein is too low to be detected by western blot.

Potential solution

Increase the total number of cells to increase the sample concentration.

Problem 5

There are more non-target stripes in western blot results.

Potential solution

Increase washing times of beads or replace the more specific antibodies.

Problem 6

Different labels may have crosstalk.

Potential solution

We provide these tags to choose from, such as Flag-tag, HA-tag, Myc-tag, Strep-Tag Ⅱ, ALFA-tag.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Sheng Li (lishengjnmc@163.com or lisheng@henu.edu.cn).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to the technical contact, Sheng Li (lishengjnmc@163.com or lisheng@henu.edu.cn) and Bo-Wen Zhang (bowenzhangbowen@163.com).

Materials availability

Plasmids used in this study are available by contacting the lead contact, Sheng Li (lishengjnmc@163.com or lisheng@henu.edu.cn).

Data and code availability

This study did not generate new data or code.