Protocol for detecting interactions between intrinsically disordered proteins and long DNA substrates by electrophoretic mobility shift assay

Summary

Intrinsically disordered regions (IDRs) of proteins leverage their structural flexibility to play important roles in numerous cellular processes including molecular recognition. Many IDRs interact with DNA, and characterizing these interactions is crucial for understanding their biological impact. Here, we present a protocol for the in vitro detection of IDR-DNA interactions using an electrophoretic mobility shift assay. We describe a radioactive-free procedure using long DNA substrates and define steps for data analysis. Altogether, this protocol facilitates reproducible and sensitive quantification of IDR-DNA interactions.

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

Graphical abstract

Highlights

• •Accessible protocol for in vitro detection of IDR-DNA interactions
• •Steps describing the preparation of non-radioactive, linearized dsDNA substrates
• •Guidance for IDR-DNA reaction setup and resolving interactions by EMSA
• •Instructions for quantification of free and IDR-bound DNA

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

Intrinsically disordered regions (IDRs) of proteins leverage their structural flexibility to play important roles in numerous cellular processes including molecular recognition. Many IDRs interact with DNA, and characterizing these interactions is crucial for understanding their biological impact. Here, we present a protocol for the in vitro detection of IDR-DNA interactions using an electrophoretic mobility shift assay. We describe a radioactive-free procedure using long DNA substrates and define steps for data analysis. Altogether, this protocol facilitates reproducible and sensitive quantification of IDR-DNA interactions.

Before you begin

• 1.Obtain DNA substrates of interest.

Note: This protocol was optimized and is described here for the use of long, single-stranded (ss) DNA substrates (>5000 nt) and linearized double-stranded (ds) DNA substrates (>2000 bp). Long DNA substrates often give improved physiological relevance over short substrates as they provide increased binding site context and can allow for cooperative binding, which may be more applicable for IDRs that interact with DNA weakly. Specifically, we use the commercially available ΦX174 virion ssDNA which exists in a predominantly circular form, and the pBluescript II SK (+) dsDNA vector (where its linearization is described in subsequent steps). This protocol should be appropriate for other ssDNA and dsDNA substrates of other lengths and structures (including shorter substrates), though alternatives have yet to be tested in this protocol.

• 2.Use standard procedures to express, purify and quantify the IDR of interest.

Note: A detailed protocol for the expression and purification of IDRs from Escherichia coli is described by Pastic et al.¹

Note: As a starting point, we recommend testing IDR concentrations of 0.01–2.5 μM in binding reactions with DNA substrates at a final concentration of 0.2 nM. Together, this offers a wide IDR to DNA molar excess range of 50:1 to 12,500:1. The advantage of using a low DNA concentration in these assays (0.2 nM) is that less protein is needed to achieve high molar excess ratios of IDR:DNA, thus being favorable if protein yield from in-house purification methods is low. A high molar excess of IDR to DNA may be needed to detect an interaction for several reasons. First, IDRs often utilize short linear interaction motifs (SLiMs) to bind DNA which typically have low affinities for nucleic acid substrates (usually in the micromolar range).^2,3 Second, agarose gel electrophoresis resolves DNA and/or proteins in a high range of mass (relative to acrylamide-based gels). Third, IDRs are classically short in length (average of 30–200 residues in humans)⁴ and, as such, they contribute only modestly to the mass of protein-DNA complexes (per unit of IDR binding). Using a significant molar excess of protein is therefore required to promote the association of numerous IDRs on each DNA molecule. This is expected to increase substantially the mass of protein-DNA complexes (relative to naked DNA), consequently enabling convenient visualization of a shift in migration following electrophoresis of the IDR-DNA complexes. To combat the common issue of protein aggregation at high concentrations (leading to protein-DNA complexes being trapped in the gel well unable to migrate in EMSA), we have optimized the EMSA buffer to promote IDR-DNA complex migration (see recipe table in materials and equipment set-up). For example, the addition of NP-40 and β-Mercaptoethanol enhance protein solubility and prevent protein aggregation, while an optimized concentration of MgCl₂ aids in stabilizing protein-DNA interactions. Though this protocol has been optimized and used successfully with IDRs ranging in size from 10 kDa to 30 kDa, we imagine that it is applicable to a wider range of IDRs with differing molecular weights.

Note: Keep in mind that buffer components in the IDR sample are carried over into EMSA reactions. To equalize this carry-over, we add appropriate amounts of IDR suspension buffer (ISB) to each EMSA reaction so that the concentration of all components in the final reaction is the same (see step-by-step method details, steps 17-18, and Table 1). The ISB recipe table in materials and equipment set-up should be adapted accordingly to reflect the buffer components of the protein of interest. We have found that the following ISB components in EMSA reactions do not adversely affect IDR-DNA interactions: ≤ 30 mM KPO₄, ≤ 180 mM NaCl, and ≤ 120 mM imidazole (Figure S1). For buffer components other than those listed above, we recommend testing their effect on IDR-DNA interactions using a titration such as the example shown in Figure S1.

Table 1.

Example EMSA set up showing volumes of working protein stocks (either 0.5 μM or 5 μM) and final protein concentrations in each binding reaction

Binding reaction	Final protein concentration per reaction (μM)	Volume of 0.5 μM IDR (μL)	Volume of 5 μM IDR (μL)	Volume of 5 μM BSA (μL)	Volume of ISB (μL)	Volume of 2× EMSA buffer (μL)
1a	0	–	–	–	12.5	12.5
2	0.1	5	–	–	7.5	12.5
3	0.15	7.5	–	–	5	12.5
4	0.2	10	–	–	2.5	12.5
5	0.25	12.5	–	–	–	12.5
6	0.3	–	1.5	–	11	12.5
7	0.4	–	2	–	10.5	12.5
8a	0	–	–	–	12.5	12.5
9b	0.4	–	–	2	10.5	12.5

The final reaction volume is 25 μL and each reaction also contains up to 12.5 μL of IDR suspension buffer (ISB) and 12.5 μL of 2× EMSA buffer (see materials and equipment set-up for buffer recipes). Each binding reaction number corresponds with their loading order and lane numbers from left to right in the example EMSA in Figure 2.

^aFree DNA (no protein) control reactions.

^bBSA control reaction.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
EcoRI-HF restriction enzyme	New England Biolabs	Cat # R3101S
Tris base	Wisent	Cat # 600-125-1K
Invitrogen UltraPure ethylenediaminetetraacetic acid, disodium salt, dihydrate (Na2EDTA·2H2O)	Fisher Scientific	Cat #15576028
Acetic acid, glacial	Fisher Scientific	Cat # 351271-500
HEPES	Fisher Scientific	Cat # BP310-500
Magnesium chloride hexahydrate	Sigma	Cat # M9272-500G
Sodium chloride	Sigma	Cat # S6753-5KG
β-Mercaptoethanol	Sigma	Cat # M3148-100ML
Potassium phosphate monobasic	Wisent	Cat # 600-078-LG
Potassium phosphate dibasic	Wisent	Cat # 600-076-LG
Imidazole	Sigma	Cat # I2399-500G
NP-40 SurfactAmps detergent solution	Thermo Fisher Scientific	Cat # 85124
Water sterile molecular biology grade (DNase, RNase & Protease-free)	Wisent	Cat # 809-115-CL
Bovine serum albumin (BSA), fraction V	Wisent	Cat # 800-095-EG
UltraPure TAE buffer, 10×	Invitrogen	Cat # 15558042
Invitrogen UltraPure agarose	Invitrogen	Cat # 16500-100
Invitrogen UltraPure LMP agarose	Invitrogen	Cat # 16520-100
Critical commercial assays
QIAquick gel extraction kit	QIAGEN	Cat # 28704
SYBR gold nucleic acid stain	Invitrogen	Cat # S11494
SYBR green I nucleic acid stain	Invitrogen	Cat # S7563
SYBR Safe DNA gel stain	Invitrogen	Cat # S33102
Gel loading dye, purple (6×)	New England Biolabs	Cat # B7024S
1 kb DNA ladder	FroggaBio	Cat # DM010-R500
GelPilot loading dye (5×)	QIAGEN	Cat # 239901
rCutSmart buffer, (10×)	New England Biolabs	Cat # B6004S
Recombinant DNA
ΦX174 virion ssDNA (5386 nt)	New England Biolabs	Cat # N3023S
EcoRI-digested pBluescript II SK (+) dsDNA (2961 bp)	Agilent	Cat # 212207
Software and algorithms
Adobe Photoshop 2024	Adobe	https://www.adobe.com/products/photoshop.html
GraphPad Prism 9.0	GraphPad Software	https://www.graphpad.com/scientific-software/prism/
Other
ThermoMixer C	Eppendorf	Cat # 5382000023
ThermoScientific Nalgene RapidFlow sterile single use bottle top filters (0.2 μM, PES membrane)	Fisher Scientific	Cat # 0974107
Basix syringe filters, PES, sterile	Fisher Scientific	Cat # 13100106
SUB15 standard submarine gel electrophoresis unit	Hoefer Inc.	Cat # SUB15
SUB15 and SUBHT comb (16 wells; 1.5 mm thickness)	Hoefer Inc.	Cat # SUB15-C1.5-16MC
SUB10 Mini-Plus submarine gel electrophoresis unit	Hoefer Inc.	Cat # SUB10
SUB10 comb (10 wells; 2 mm thickness)	Hoefer Inc.	Cat # SUB10-C2-10MC
PS300B 300 Volt power supply	Hoefer Inc.	Cat # PS300B
D-DiGit gel scanner unit	LICORbio	Cat # 3500-00
NanoDrop One microvolume UV spectrophotometer	Thermo Fisher Scientific	Cat # ND-ONE-W
ProFlex 3 × 32-well PCR system	Thermo Fisher Scientific	Cat # 4484073
BrandTech PCR 8-strip tubes	Fisher Scientific	Cat # 14380942
1.5 mL Eppendorf tubes	Fisher Scientific	Cat # 14-666-319
Eppendorf 5424 R Microcentrifuge	Eppendorf	Cat # EP5404000138
Eppendorf Rotor F45-24-11	Eppendorf	Cat # EP022653008
Eppendorf Rotor F45-32-5-PCR	Eppendorf	Cat # EP5424704000
Fisherbrand Isotemp digital dry baths/block heaters	Fisher Scientific	Cat # 88860022

Materials and equipment

Alternatives: While we highly recommend using a PCR machine to maintain constant temperature during the IDR-DNA binding reaction step, we recognize that this equipment is not always available. Incubating reactions in 1.5 mL Eppendorf tubes in a standard heat block should provide satisfactory results, but we have not tested this alternative. Optimization steps may be needed.

Alternatives: We use a dry, orbital shaking/heating block (Eppendorf Thermomixer C) to promote uniform mixing of a 1.6% LMP agarose solution at 500 rpm while maintaining its temperature at 33°C. If this equipment is not available, it may be possible to use a standard heat block affixed to an orbital shaker.

Alternatives: This protocol uses a D-DiGit gel scanner unit to visualize SYBR Safe-stained DNA in the agarose gel. Alternative gel imaging systems with a compatible DNA stain should be applicable in place of the D-DiGit. If using a UV-based transilluminator with an ethidium bromide-stained gel, ensure appropriate precautions are taken to minimize UV and EtBr exposure. The advantage of the D-DiGit is that it uses a non-UV light source which coupled with the SYBR Safe DNA stains makes for a safe and highly sensitive detection system.

Note: This protocol is sensitive to contaminants and precipitates. Unless otherwise indicated, all solutions are filter sterilized using a 0.2 μM PES membrane filter.

50× TAE buffer

Reagent	Final 50× concentration	Stock concentration	For 1L of 50× TAE
Tris base	2 M	–	242 g
Acetic acid	5.71% (v/v)	100% (v/v)	57.1 mL
EDTA (Na2EDTA·2H2O) pH 8.0	50 mM	0.5 M	100 mL
MilliQ H2O	–	–	Up to 1 L

Adjust the pH to 8.0 if necessary. Filter sterilize the solution and store at 20°C–22°C for 6 months.

Alternatives: TAE buffer is also commercially available as a sterile-filtered solution. For example, Invitrogen supplies UltraPure TAE Buffer, 10×. We use the commercial buffer to prepare the 1% agarose gel for EMSA analysis (see step-by-step method details, step 13). The recipe above for in-house 50× TAE is used to prepare running buffer and gel staining buffer (see step-by-step method details, step 14 and 33D respectively), as well as for the 1% agarose gel used for linearized dsDNA substrate isolation (see step-by-step method details, step 3).

2× EMSA buffer

Reagent	Final 2× concentration	Stock concentration	Per 25 μL reaction	Master mix for ∼17 × 25 μL rxnsa
HEPES pH 7.5	20 mM	0.5 M	0.5 μL	8.5 μL
MgCl2	14 mM	0.5 M	0.35 μL	5.95 μL
NaClb	300 mM	0.5 M	7.5 μL	127.5 μL
β-Mercaptoethanolb	4 mM	25 mM	2 μL	34 μL
NP-40	1% (v/v)	10% (v/v)	1.25 μL	21.25 μL
DNA substrate	0.4 nM	12.5 nM	0.4 μL	6.8 μL
Nuclease-free water	–	–	Up to 12.5 μL	Up to 212.5 μL

Filter sterilize each stock solution except for β-Mercaptoethanol, NP-40 and the DNA substrate. Make up a fresh master mix of EMSA buffer immediately prior to each assay and keep on ice until use. This buffer cannot be stored and reused as β-Mercaptoethanol is unstable.

^aThough the agarose gel used in this protocol can accommodate 16 reactions, make up enough EMSA buffer for 17 reactions to account for pipetting error.

^bCustom adjustments may need to be made to the concentrations of NaCl and β-Mercaptoethanol in the 2× EMSA recipe depending on whether your IDR storage buffer contains them (these reagents are commonly used in protein storage buffers). For example, our storage buffer contains 300 mM NaCl and 2 mM β-Mercaptoethanol. Since half of the 25 μL EMSA reaction volume is IDR suspension buffer (see step-by-step method details, step 17–18), this carries over sufficient NaCl and half of the required β-Mercaptoethanol concentration into the binding reactions. Thus, we omit NaCl from our 2× EMSA buffer, and only add 2 mM β-Mercaptoethanol (this is not reflected in the buffer recipe above).

Example IDR suspension buffer (ISB)^a

Reagent	Final concentration	Stock concentration	For 1 mL of ISB
KPO4 pH 8.0	50 mM	1 M	50 μL
NaCl	300 mM	0.5 M	600 μL
Imidazole	200 mM	1 M	200 μL
β-Mercaptoethanol	2 mM	25 mM	80 μL
Nuclease-free water	–	–	70 μL

Make ISB fresh immediately before each experiment, filter sterilize and keep on ice until use. Do not store and reuse as β-Mercaptoethanol is unstable.

^aThis is simply an example of an ISB recipe and reflects the concentrations of each reagent of the buffer that our specific proteins are stored in. Modifications to this recipe should be made depending on the storage buffer components of the protein to be used in this assay. For example, if a component in the storage buffer is 10% glycerol, the ISB should also contain 10% glycerol. As previously mentioned in before you begin: Note 2, buffer components that are not listed in this table should be tested in EMSA reactions first to ensure that they do not have adverse effects on IDR-DNA binding (see Figure S1).

Step-by-step method details

Linearization of dsDNA substrates—Day 0

Timing: 4–5 h

In this step, circularized dsDNA is linearized for EMSA application.

• 1.
  Set up a pBluescript II SK (+) vector digestion reaction using EcoRI.

  • a.
    In a PCR tube, mix the following reagents:

    • i.
      500 ng pBluescript II SK (+) vector.
    • ii.
      4 μL rCutSmart buffer (10×).
    • iii.
      10 U EcoRI-HF enzyme.
    • iv.
      X μL of filter sterilized MilliQ H₂O (up to 40 μL).
  • b.
    Mix the contents by gently flicking the tube.
  • c.
    Briefly centrifuge (5–10 sec at maximum speed) to collect all contents at the bottom of the tube.
• 2.Incubate the reaction(s) at 37°C for 2 h in a PCR block.

Alternatives: Plasmid digestion can also be performed in a 1.5 mL Eppendorf tube using a standard heat block.

Note: We suggest setting up multiple reactions at the same time to obtain more DNA.

• 3.
  During the 2 h digestion period, prepare a 1% agarose gel for DNA extraction.

  • a.
    Combine 1 g of UltraPure agarose with 100 mL of 1× TAE in a 250 mL Erlenmeyer flask.
  • b.
    Microwave the agarose mixture for 45 sec. Swirl the flask to mix, then microwave an additional 30 sec (or until the agarose has fully dissolved).
  • c.
    Let the agarose mixture cool at 20°C–22°C (room temperature) for 10 min.
  • d.
    Add 10 μL of SYBR Safe DNA gel stain and swirl the flask to mix.
  • e.
    Pour the entire agarose solution into a 10 × 11.5 cm gel tray and add a 10-well, 2 mm comb.
  • f.
    Once solidified, place the gel tray into the electrophoresis unit and fill the unit with 1× TAE.

Alternatives: We use the SUB10 Mini-Plus submarine gel electrophoresis unit for step 3, but other electrophoresis units can be used for this step. Agarose gel volume may need to be adjusted accordingly depending on the size of the gel tray. Ideal gel thickness should be approximately 0.8–1 cm.

CRITICAL: Though in-house or kit-based PCR purification is another method of DNA isolation, this is not recommended as it will not remove undigested product.

• 4.
  Once the incubation period in step 2 is complete, remove the digestion reaction(s) from the PCR machine and add 8 μL of 6× gel loading dye.

  • a.
    Mix the contents by pipetting up and down.
• 5.Remove the comb from the agarose gel prepared in step 3 and load 5 μL of a 1 kb DNA ladder in one lane of the gel.
• 6.Load the digested product equally into 3 wells of the gel (∼16 μL per well).
• 7.Perform electrophoresis for 60 min at 100 V, or until the red dye front reaches at least halfway through the gel.
• 8.Stop the electrophoresis and visualize the DNA using the D-DiGit gel scanner unit.

Note: A prominent DNA band should appear at approximately 3 kb (Figure 1). Bands at ∼2.5 kb or ∼4.5 kb could signify undigested plasmid that remains supercoiled or nicked plasmid respectively (Figure 1). For assistance resolving this issue, see troubleshooting 1.

• 9.Excise the DNA band of interest from each lane using a clean razor and place each band into a separate 1.5 mL Eppendorf tube.

Note: We have found that splitting the product into multiple tubes for extraction increases DNA recovery efficiency.

Pause point: Gel slices containing DNA can be stored at 4°C in Eppendorf tubes sealed with parafilm for at least 2 weeks.

• 10.
  Use the QIAquick gel extraction kit (Qiagen) to recover DNA from the gel slices according to manufacturer’s instructions with a few exceptions noted below (website: https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/dna-clean-up/qiaquick-gel-extraction-kit).

  • a.
    Use a separate column for each gel slice to enhance DNA yield.
  • b.
    Elute DNA from each column in 10 μL of nuclease-free water.

    Note: The elution buffer provided in the QIAquick kit (1× TE) is not explicitly labeled as nuclease-free, thus we recommend using nuclease-free water for DNA elution instead.

  • c.
    Combine eluted DNA fragments into one Eppendorf tube and determine the concentration.

    Alternatives: Instead of using a kit, recovery of DNA from the agarose gel slices can be performed using in-house methods.⁵ However, this has not been tested alongside this protocol.

• 11.Dilute the DNA to a working concentration of 12.5 nM with nuclease-free water.
• 12.Aliquot diluted DNA stocks into 20 μL volumes and store at −20°C.

Pause point: Linearized dsDNA aliquots can be stored at −20°C for up to 6 months.

Note: If you also plan to use the ΦX174 virion ssDNA substrate in EMSA, perform steps 11–12 to generate diluted aliquots of 12.5 nM from the main stock. Diluted aliquots can be stored at −20°C for up to 6 months. Undiluted stocks can be stored at −20°C or −80°C for 2–3 years.

CRITICAL: Aliquoting the diluted stock into smaller volumes prevents DNA degradation by reducing the number of freeze-thaw cycles sustained. We suggest that each aliquot endure a maximum of two freeze-thaw cycles.

Figure 1.

Migration pattern of EcoRI-digested or undigested pBluscript II SK(+) vector (2961 bp) in a 1% agarose gel

EcoRI-digested vector (blue box) is excised from the gel and purified to be used as a linear dsDNA substrate for EMSA.

Electrophoretic mobility shift assay—Day 1

Timing: 20 h (4 h of reaction preparation/incubation and 16 h of electrophoresis)

In this step, the IDR of interest is incubated with potential DNA substrates. Electrophoresis is used to separate IDR-bound DNA from free DNA on an agarose gel.^6,7

• 13.
  Prepare a 1% agarose gel.

  • a.
    Add 1.5 g of UltraPure agarose to 150 mL 1× TAE in a 250 mL Erlenmeyer flask.

    Note: As previously mentioned, we use commercially available 10× UltraPure TAE buffer (see key resources table) for agarose gel preparation to minimize variability between replicates. 50× TAE prepared in-house can also be used but may affect reproducibility or cause DNA bands to smear if prepared incorrectly (see troubleshooting 4).

  • b.
    Microwave the mixture for 1 min and swirl the contents to ensure uniform heating.
  • c.
    Microwave an additional 30 sec.
  • d.
    Check to ensure that the agarose has fully dissolved (it should not have any solid precipitates).
  • e.
    If not fully dissolved, microwave in 10 sec increments until no solid precipitates remain and the agarose has completely dissolved.

    CRITICAL: Keep a close eye on the agarose mixture during the microwaving steps. Do not let it boil over the top of the flask as any agarose loss will affect the final gel volume/concentration, and this interferes with the reproducibility of the experiment (see troubleshooting 4).

  • f.
    Let the agarose mixture cool at 20°C–22°C for approximately 10 min.

    Optional: The above step is not necessary for the integrity of the assay but will prolong the lifespan of the gel tray. We have found that pouring boiling agarose into the gel trays causes them to crack.

  • g.
    Meanwhile, clean a 15 × 15 cm gel tray, casting gates, and a 16-well, 1.5 mm gel comb with 70% ethanol.
  • h.
    Rinse the tray, gates and comb with filtered MilliQ H₂O and allow them to air dry until the agarose is ready to be poured.

    CRITICAL: This step will help to remove any contaminating nucleic acids from previous uses and prevent non-specific signal from being detected in the gel later. (See troubleshooting 6).

  • i.
    Once cooled, pour the entire agarose mixture into the gel tray and place the comb into the top of the gel.

    Note: Though a gel tray with different dimensions and comb measurements could be used for this step, we recommend using those described above for the purpose of protocol reproducibility. If a larger or smaller gel tray is used, agarose gel preparation will need to be optimized accordingly. The gel should be approximately 0.8–1 cm thick.

  • j.
    Allow the gel to solidify at 20°C–22°C, then put it at 4°C for at least 2–3 h. EMSA reactions can be prepared during this time.

    CRITICAL: Keep the gel in a covered, isolated area during solidification to prevent dust or dirt particles from entering it.

• 14.Prepare 1 L of filter sterilized 1× TAE to use as a running buffer during electrophoresis and keep it at 4°C.
• 15.Thaw IDR samples of interest and working stocks of DNA substrates on ice and pre-cool a microcentrifuge to 4°C.

Alternatives: If a microcentrifuge with a cooling function (such as the Eppendorf 5424 R) is not available, a standard microcentrifuge can be operated in a 4°C room. We suggest bringing the microcentrifuge to the 4°C room at least 1 h prior to its use to allow it to cool down.

CRITICAL: Keep IDR and DNA samples on ice throughout this protocol to avoid degradation (see troubleshooting 3).

• 16.Once thawed, gently flick each tube to mix (≥ 10 times) and briefly centrifuge to collect all liquid at the bottom of the tube (5–10 sec at 15,000 × g) in the pre-cooled microcentrifuge.
• 17.
  Prepare the following EMSA reagents in 1.5 mL Eppendorf tubes and keep on ice:

  • a.
    Make a master mix of 2× EMSA buffer containing the DNA substrate of choice (linearized dsDNA prepared in steps 1–12 or ssDNA).
  • b.
    Prepare 1 mL of IDR suspension buffer (ISB).
  • c.
    If testing the recommended IDR concentration range of 0.01–2.5 μM, make up two working dilutions of the IDR of interest in ISB:

    • i.
      0.5 μM (i.e., to test concentrations of 0.01–0.25 μM).
    • ii.
      5 μM (i.e., to test concentrations of 0.25–2.5 μM).

      Alternatives: Once these ranges have been tested, they can be adjusted accordingly for concentrations of interest.

      Note: Since there are 16 lanes available, we recommend choosing 10–12 IDR concentrations to test against DNA substrates. The other 4 lanes will be used for a negative control protein (see the next step and 18D), and for free DNA migration (see ‘Critical’ note in step 18A).

  • d.
    Prepare a 5 μM solution of Bovine Serum Albumin (BSA) in ISB to be used as a negative control for DNA-binding.
  • e.
    Mix all solutions well by flicking the tube followed by a brief centrifugation in the pre-cooled microcentrifuge (as performed in step 16) to collect the liquid at the bottom of the tube.
• 18.
  Prepare EMSA reactions for each protein concentration in PCR tubes on ice:

  • a.
    Label the desired number of PCR tubes with the IDR concentration and DNA substrate identity.

    CRITICAL: Include 2–3 tubes as free DNA control reactions that will not receive the IDR of interest. These reactions are critical for demonstrating the migration of free DNA during electrophoresis. Also include 1 tube to receive the negative control protein (BSA).

  • b.
    Aliquot 12.5 μL of 2× EMSA buffer into each PCR tube.
  • c.
    Add the desired volume of 0.5 μM or 5 μM diluted IDR stocks to the appropriate tubes to reach the protein concentration of interest.
  • d.
    Add 12.5 μL of 5 μM BSA (or the highest equivalent volume/concentration as the protein of interest) to the tube set aside for the negative control.
  • e.
    Add ISB to all tubes (including controls) to a final reaction volume of 25 μL.
  • f.
    Flick the tubes to mix the contents well (≥ 10 times).
  • g.
    Briefly spin the tubes in a pre-cooled microcentrifuge (as performed in step 16) equipped with a PCR strip tube rotor to collect all liquid at the bottom of the tube.

    Alternatives: If a cooling microcentrifuge or PCR strip tube rotor is not available, a portable mini-centrifuge equipped with a PCR tube adaptor can be operated in a 4°C room (such as the Fisherbrand Mini-centrifuge, Cat # 12-006-901).

    Note: For an example reaction set up and gel loading order, see Table 1. The corresponding EMSA gel is shown in Figure 2. Free DNA control reactions (no protein) are typically loaded in the left-most and right-most wells of the gel to control for inconsistencies in DNA migration.

• 19.Quickly bring the tubes containing all binding reactions to a PCR block and incubate the reactions at 30°C for 35 min. Set the PCR block to cool to 4°C after the incubation is finished.

CRITICAL: Proceed immediately with step 22 once the incubation has finished. This will reduce the opportunity for protein degradation prior to electrophoresis (see troubleshooting 3).

• 20.Meanwhile, set a heat block to 95°C and set the Eppendorf Thermomixer C (or alternative shaking-incubating apparatus) to 33°C.

Note: Ensure that there is enough bench space beside the thermomixer to accommodate your 1% agarose gel. If not, move it to a location where you will have enough space to put your gel beside it. It is important to load your gel while having immediate and unrestricted access to the thermomixer.

• 21.
  Prepare 3 × 1 mL 1.6% Low Melting Point (LMP) agarose in 1.5 mL Eppendorf tubes.

  Note: LMP agarose will eventually be added to each binding reaction prior to loading them on the agarose gel. The purpose of this is to seal the reactions inside the wells to minimize sample loss once the gel is submerged in running buffer. The addition of LMP agarose to binding reactions also protects long DNA substrates against shearing.

  • a.
    For each agarose aliquot, weigh out 16 mg of LMP agarose.

    Note: We find that the simplest way to obtain exactly 16 mg of LMP agarose in a reproducible manner is to weigh the agarose in the Eppendorf tube rather than in a weigh boat. This way, you can be sure that you won’t lose any during its transfer to the tube.

  • b.
    Add 1 mL of filter sterilized 1× TAE to each tube and vortex vigorously for 10 sec.

    CRITICAL: Before proceeding, ensure that the agarose is evenly mixed and not clumped in the bottom of the tube. If clumps are present, use a pipette tip to dislodge them and repeat the prior vortex step. Homogenous mixing of the LMP agarose is critical to avoid uneven gel polymerization.

  • c.
    Dissolve the LMP agarose at 95°C in the heat block for 5 min.

    Alternatives: If you are not using Eppendorf tubes with a lid-locking mechanism, tape the caps of the tubes down to prevent them from bursting open during the boiling step.

  • d.
    After 5 min, remove the LMP agarose from the heat block and shake vigorously at least 10 times to ensure even distribution of agarose prior to the cooling step.

    Note: If the caps are taped down, tape can be removed after this step.

  • e.
    Quickly, bring the tubes of agarose to the thermomixer and incubate at 33°C with 500 rpm orbital shaking.

    Note: LMP agarose tubes will remain in the thermomixer until the binding reactions are ready to load on the 1% agarose gel.

    CRITICAL: It is important to follow these steps precisely. If prepared incorrectly, this can cause DNA bands to be smeared or streaked during electrophoresis (see troubleshooting 4).

• 22.
  Once the binding reactions have completed their incubation, add 1 μL of GelPilot loading dye (5×).

  • a.
    Mix gently by pipetting up and down.

CRITICAL: Avoid harsh methods of mechanical mixing after reaction incubation (i.e., vortexing). Vortexing has been suggested to cause irreversible dissociation of protein-DNA complexes.⁶ (See troubleshooting 3).

• 23.Bring the binding reactions and the 1% agarose gel (prepared in step 13) to the benchtop beside the thermomixer containing the tubes of LMP agarose.
• 24.Gently remove the comb from the agarose gel.
• 25.Stop the thermomixer from shaking (but keep the temperature at 33°C).

Note: The LMP agarose should be slightly viscous but still liquified. Mix by inverting the tube several times prior to using each aliquot.

• 26.Add 25 μL of LMP agarose to the first binding reaction and pipette up and down to mix evenly (i.e., until the dye is homogenously distributed). Immediately load the sample into the first well of the agarose gel.
• 27.Repeat this process with the remainder of the binding reactions. Load the second free DNA control and BSA negative control reactions last.

CRITICAL: It is imperative to load each reaction immediately after mixing it with the LMP agarose as it will begin to solidify in a matter of seconds. Work quickly, but also carefully to avoid losing any of the sample. Ensure that the entire sample is loaded into the well. The consequences of pipetting errors are described in troubleshooting 5.

Optional: We find it useful to rest the pipette tip gently against one of the sides of the wells while loading the samples.

Note: We have not noticed any undesired effects of air bubbles in the sample on binding results or gel image quality.

• 28.Once all reactions have been loaded, add the remaining LMP agarose to the top of all wells to seal the contents inside.

Note: Be generous and ensure that the wells are fully covered.

• 29.Put the agarose gel at 4°C for 15 min to allow the LMP agarose to fully solidify within the wells.
• 30.
  Meanwhile, set up the SUB15 gel electrophoresis unit and a PS300B 300 V power supply unit in a 4°C cold room.

  • a.
    Pour 1 L of cold 1× TAE prepared in step 14 into the tank.
• 31.Once the LMP agarose has solidified, remove the casting gates at either end of the gel tray and place the gel into the SUB15 electrophoresis unit (or alternative apparatus) allowing it to be fully covered with 1× TAE.

Note: You do not need to remove the gel from the tray at this step (it will be removed during staining in step 34).

• 32.Perform electrophoresis for 16 h at 40 V.

Figure 2.

An example outcome of an EMSA with 0–0.4 μM IDR protein and 0.2 nM ssDNA substrate (ΦX174)

(A) Free DNA was separated from IDR-bound DNA on a 1% agarose gel. A prominent upwards band shift occurs when IDR-DNA complexes are formed. Bovine serum albumin (BSA) was combined with ssDNA substrates at the highest corresponding concentration to the IDR of interest (0.4 μM) as a negative control for DNA-binding. Lane numbers are specified at the bottom of the gel.

(B) IDR-bound DNA in lanes 2–7 from panel (A) was quantified relative to the sample that contained no protein (lane 1) in the EMSA gel. Data was fit with a Hill-Langmuir equation and a dissociation constant (K_D) was obtained from this curve. K_D describes the protein concentration at which 50% DNA is bound. Data represents the replicate shown in panel A.

Staining the gel—Day 2

Timing: 3 h

In this step, we will stain the agarose gel and visualize the DNA bands using an LED-based gel imaging system.

• 33.
  Prepare a light-protected container to stain the agarose gel.

  Note: We use a plastic container covered in aluminum foil.

  • a.
    Clean the inside of the container with 70% ethanol to remove any nucleic acids or other precipitates that could contaminate the gel.
  • b.
    Rinse the container with filtered MilliQ H₂O and allow it to dry on the bench top.
  • c.
    Meanwhile, thaw a vial of SYBR nucleic acid stain (Green I for dsDNA, Gold for ssDNA) at 20°C–22°C protected from light.

    Note: Pre-aliquot this stain in 25 μL volumes to avoid multiple freeze thaw cycles. Store it at −20°C protected from light.

    Note: Though SYBR gel stains are less hazardous staining reagents than others (like ethidium bromide), we still recommend exercising caution. Always wear PPE when handling the stain and manipulating SYBR stained gels.

  • d.
    Prepare 800 mL of 1× TAE and put 200 mL into the light-protected container. The remaining 600 mL can be kept aside at 20°C–22°C.
  • e.
    Once the stain has thawed, add 25 μL to the 200 mL 1× TAE in the light-protected container.
  • f.
    Immediately replace the lid and swirl the solution for 30 sec to mix.
• 34.Carefully, remove the agarose gel from the electrophoresis unit.
• 35.Place the gel into the staining container and incubate it on an orbital shaker at 100 rpm for 1 h 15 min at 20°C–22°C.
• 36.Remove the stain and wash the gel in 3 successive cycles with 200 mL of 1× TAE for 10 min each on the orbital shaker at 100 rpm.
• 37.Visualize the gel using the D-DiGit gel scanner unit.

Note: We recommend using each of the pre-set exposures (from 4×–24×) to capture at least one quantifiable image (see quantification and statistical analysis, step 1).

Expected outcomes

Agarose gel electrophoresis will separate free DNA from IDR-bound DNA based on the observation that the electrophoretic mobility of nucleic acids is reduced when bound by protein.^6,7,8,9 Upon imaging the gel, you should anticipate seeing one sharp band in the free DNA control lanes that represents the electrophoretic migration distance of unbound DNA (see ‘Free DNA’ in lanes 1 and 8 in Figure 2A). You should also observe a band at this height in the negative control lane as BSA has not been found to bind DNA using this protocol¹ (see BSA in lane 9 in Figure 2A). If the IDR of interest interacts with the DNA target, you should expect to see DNA bands beginning to shift upwards with increasing protein concentration in a ladder-like formation (in other words, the migration distance shortens due to increased electrophoretic inhibition). This is referred to as a ‘band shift’ and is an indication of IDR-DNA complex formation (Figure 2A). In all cases, DNA bands should be distinct and sharp. If bands are diffuse, smeared, or stuck in the well, see the troubleshooting guide. When a certain molar excess of IDR to DNA is reached, 100% of DNA will be bound by protein and no free DNA will remain at the bottom of the lane. The exact protein concentration at which this occurs can be identified by quantification of the free DNA in each lane as described in quantification and statistical analysis.

Quantification and statistical analysis

Standard image analysis programs including Fiji and Adobe Photoshop can be used to process and analyze an EMSA. Here, we describe a precise set of steps to estimate the fraction of free DNA in each EMSA binding reaction using Adobe Photoshop. GraphPad Prism or an alternative data processing software can then be used to plot the data and obtain an approximate dissociation constant (K_D) representing the concentration of protein in which 50% of the DNA substrate is bound (see an example in Figure 2B).

• 1.Open the gel image file of interest with Adobe Photoshop. We recommend choosing an image where the bands are sharp but not overexposed (for example, the gel image in Figure 2A).
• 2.Invert pixel intensities and adjust the brightness and contrast to reduce the background signal (Figure 3A).

CRITICAL: Only use the “slider tool” to adjust pixel brightness and contrast (shown by a white arrow in Figure 3A). Do not use image processing functions that modify image data in a non-linear manner.

• 3.
  Align the free DNA bands horizontally (in lanes with no protein).

  • a.
    Drag a guide line from the horizontal ruler (Guide #1 in Figure 3B) so that it divides the first free DNA band in half on a horizontal plane (lane 1 in Figure 3B).
  • b.
    If necessary, rotate the gel so that second free DNA band (lane 8 in Figure 3C) is also equidistantly split on the horizontal plane by Guide #1.
  • c.
    Lock Guide #1 in place for the remainder of the steps. This guide represents the baseline of free DNA migration based on lanes 1 and 8.
• 4.
  Use the marquee and histogram tools along with Guide #1 to determine relative quantity of the free DNA in each lane.

  • a.
    Draw a rectangular marquee box to fit precisely around the free DNA band in lane 1 (Figure 3D).

    Note: Guide #1 should run through the exact middle of this box. If it doesn’t, you may need to repeat step 3 to properly align the gel.

  • b.
    Open the histogram tool and record the mean pixel intensity within the box in lane 1 (Figure 3D).
  • c.
    Use the right keyboard arrow to move the same marquee box horizontally along Guide #1 to each of the other lanes (Figure 3E) and record the mean pixel intensity of free DNA in each one. You should anticipate that this value will decrease as IDR-DNA complexes form and the DNA band begins to shift upwards.

    CRITICAL: Guide #1 must remain in the middle of the marquee box during this process (i.e., avoid moving the box using the up and down keyboard arrows or the keypad/mouse).

• 5.
  Correct the free DNA intensity measurements by subtracting background pixel intensity.

  • a.
    Drag a second horizontal guide 1–2 cm underneath the free DNA bands (Guide #2 in Figure 3F).
  • b.
    Use the same marquee box from step 4 and the histogram tool to record the background pixel intensity for each lane (Figure 3G).
  • c.
    Like in step 4B, move the marquee box horizontally along Guide #2.
  • d.
    For each lane, subtract the background intensity from free DNA intensity to obtain a corrected free DNA intensity value (Figure 3H).
• 6.
  Estimate the fraction of bound DNA in each sample based on corrected free DNA values.

  • a.
    For each individual sample, multiply the corrected free DNA intensity value by 100.
  • b.
    Divide by the corrected free DNA intensity of the no protein sample (i.e., lane 1). This will give you the approximate percentage of free DNA in that sample.
    For example (from Figure 3H):

    $$\%\text{free}\text{DNA}\text{in}\text{lane}4=(100\ast \text{free}\text{DNA}\text{intensity}\text{lane}4)/\text{free}\text{DNA}\text{intensity}\text{lane}1$$

    $$=(100\ast 22.67)/170.05$$

    $$=13.33\%$$
  • c.
    The approximate fraction of IDR-bound DNA in each fraction can be obtained by subtracting the percentage of free DNA from 100.
    For example:

    $$\%\text{IDR}-\text{bound}\text{DNA}\text{in}\text{lane}4=100–\%\text{free}\text{DNA}$$

    $$=100–13.33$$

    $$=86.67\%$$
• 7.
  Determine the K_D of the IDR-DNA interaction.

  • a.
    Use GraphPad Prism (or an alternative data analysis software) to plot the percentage of IDR-bound DNA relative to the protein concentration in each sample (see the example in Figure 2B).
  • b.
    Determine the most appropriate model (equation) to fit your data.

Note: This will largely depend on the IDR-DNA interaction kinetics but will typically be non-linear. GraphPad Prism offers many built-in non-linear regression models which will offer an accompanying data sheet that includes an approximate K_D. For more information on curve fitting using GraphPad, see website: http://www.graphpad.com/guides/prism/latest/curve-fitting/index.htm

Note: For estimation of K_D, we recommend performing ≥ 3 replicates. If comparing the DNA binding affinity of two proteins against each other, we strongly suggest running the samples on the same gel.

Note: As a general guideline, we omit gels from our analysis when DNA bands are stuck in the wells (Figure 4A), are indistinct/smeared or show an irregular migration pattern (Figures 4B–4D), or when there is non-specific signal that interferes with the quantification process (Figure 4E).

Figure 3.

Step-by-step description of EMSA quantification and analysis using Adobe Photoshop

(A) The gel file of interest is opened in Adobe Photoshop. The image is inverted, and brightness and contrast are uniformly adjusted.

(B) Guide line #1 (cyan) is dragged down from the horizontal ruler tool and aligned in the middle of the DNA band in the left-most free DNA (no protein) control sample (lane 1).

(C) The gel is rotated so that Guide #1 also intersects the DNA band in the right-most free DNA control (lane 8).

(D) A rectangular marquee box (cyan) is drawn around the free DNA band in lane 1 and is intersected in its middle by Guide #1. Using the histogram function, the average pixel intensity within the marquee box is recorded which represents free DNA intensity in the no protein sample in lane 1.

(E) The rectangular box is then moved horizontally along Guide #1 to record the pixel intensity of the free DNA band in each consecutive lane.

(F) A second guide (yellow) is pulled down from the horizontal ruler and placed just underneath Guide #1. This serves as the background pixel intensity guide.

(G) Using the same rectangular marquee box as panel (D) (shown in yellow) with the histogram tool, the average background pixel intensity of each lane is documented. As performed in panel (E), the box is moved horizontally along Guide #2.

(H) Background pixel intensity is subtracted from free DNA pixel intensity for each lane to obtain a corrected free DNA intensity value. This value can then be used to determine the fraction of free DNA and IDR-bound DNA in each sample (see quantification and statistical analysis, step 6).

Figure 4.

Representations of unexpected or undesired EMSA outcomes

(A) DNA migration is unobstructed in the reactions with no IDR, but upon addition of the IDR, DNA becomes trapped in the wells.

(B) IDR-DNA complex bands are broad and indistinct, while free DNA (in reactions with no IDR and BSA control) are sharp and show expected migration patterns.

(C) All DNA bands show a smeared or streaked pattern.

(D) DNA bands are sharp but show an irregular migration pattern.

(E) Agarose gel contains non-specific or background signals that interfere with quantification and data analysis.

(F) No band shift is detected in any reactions. In all panels, free DNA was separated from IDR-DNA complexes by electrophoresis on a 1% agarose gel. Potential solutions to these undesired outcomes are discussed in the troubleshooting section.

Limitations

One of the most prominent limitations of the EMSA with disordered protein regions is the propensity of some IDRs to aggregate and/or precipitate. Protein aggregation often causes DNA to be trapped in the wells of the agarose gel during electrophoresis. We have found that adding a non-ionic detergent into the EMSA reactions (NP-40) aids in mitigating IDR aggregation and allows for sample migration. Additionally, we have observed that IDR precipitation is more likely to occur in protein samples with a concentration of 50 μM or greater (or equivalent metric measurement in mg/mL based on IDR molecular weight). This is amplified if centrifugal methods with membrane-based filters are used to further concentrate a purified protein. We recommend exercising caution if these methods are to be performed. A second, important consideration of the IDR-based EMSA is that an IDR is often only a small section of a much larger, globular protein. Though this assay provides valuable insight into the DNA-binding capacity of IDRs, it is difficult to conclude the physiological relevance of an IDR-DNA interaction in the context of the entire protein. Additional in vivo experiments could be performed to determine the significance of an IDR-DNA reaction, for example, using chromatin immunoprecipitation with the full-length protein and a variant missing its IDR. Finally, the EMSA alone provides little information about the location of an IDR-binding site within a DNA substrate. The long DNA substrates used in this protocol could contain hundreds of possible nucleic acid sequences occupied by the IDR of interest, but the identity of these sequences is not discernable using this assay. This information can instead be gained using well-established nuclease or chemical footprinting assays^10,11 or using modern methods such as high-throughput sequencing SELEX.¹²

Troubleshooting

Problem 1

Incomplete digestion of plasmid DNA (for the creation of linearized substrates in step-by-step method details, step 1). See Figure 1 for an example of undigested plasmid.

Potential solution

This suggests that the restriction enzyme is not working at 100% capacity, or that the digestion conditions are unfavorable.

• •Check the expiry date of the restriction enzyme. Obtain a new enzyme if necessary.
• •Increase the suggested incubation time (2 h) to 4 h or 18 h (overnight), but monitor for Star activity (non-specific cleavage).
• •Use high-fidelity (HF) enzymes if longer incubations are needed (HF enzymes are engineered to reduce Star activity).
• •If using a different vector than the one suggested in this protocol, try decreasing the amount of vector in the digestion reaction (i.e., 200 ng instead of 500 ng). In our experience, some vectors are more difficult to digest than others, perhaps due to the formation of secondary structures. Decreasing the amount of vector in the reaction often improves digestion efficiency.
• •Ensure that a compatible buffer is being used for the restriction enzyme (i.e., rCutsmart for EcoRI-HF). If using a different restriction enzyme, see the buffer compatibility table supplied on the NEB website: https://www.neb.com/en/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes?srsltid=AfmBOooqfbEoctOkLqgBMn3qZ-xoe0F93P1aWzuXsbTJ4dlfb267Se7G.

Problem 2

DNA migrates normally in the absence of IDR (and when combined with BSA) but gets trapped in the wells when incubated with the IDR (see the example in Figure 4A).

Potential solution

It is likely that the IDR sample contains unwanted contaminants or is aggregating/precipitating, thereby preventing the DNA from entering the gel during electrophoresis in step-by-step method details, step 32. Be aware that this is not necessarily an indication of protein-DNA complex formation and may simply be an artifact of protein aggregation.

• •If the IDR was purified using a large affinity tag like GST, this tag should be cleaved and removed from the IDR prior to its use in EMSA. GST is known to undergo dimerization and thus will promote aggregation of GST-tagged proteins.¹³ Small affinity tags such as hexahistidine (6×His) have not incited protein aggregation in our experience and can be left uncleaved if desired.
• •Ensure that your protein sample is at least 95% pure and does not contain large molecular weight contaminants. The sample may need to be re-purified using more stringent conditions if the issue persists. An effective and robust IDR purification protocol is described by Pastic et al.¹
• •Check the purified IDR sample for detectable precipitates (either by eye or using a microscope). To remove precipitated protein, samples can be centrifuged in an Eppendorf Centrifuge 5424 R at 13,000 × g for 30 min at 4°C. The supernatant can be taken to a new tube and pellet/precipitates can be discarded. Keep in mind that you may need to re-quantify your protein sample after removing aggregates.
• •Increase the concentration of NP-40 in the 2× EMSA buffer by 1%–2% (adjust from the recipe in materials and equipment setup, 2× EMSA buffer). NP-40 is a mild, nonionic detergent that supports protein solubility. The final concentration of NP-40 in EMSA reactions in this protocol is 0.5%, but we have also successfully tested 1%. Alternatively, a different detergent can be tested in place of NP-40 depending on the identity of the IDR. For example, the zwitterionic detergent CHAPS provides higher solubilization for hydrophobic proteins.¹⁴
• •The protein to DNA ratio could be too high. Try testing a lower range of protein concentrations than those suggested in step-by-step method details, step 17C.

Problem 3

Free DNA (and BSA control) bands are sharp, but IDR-DNA complex bands are broad and indistinct without being trapped in the wells (see the example in Figure 4B).

Potential solution

This suggests that IDR-DNA complexes have partially dissociated at some point before or during the electrophoresis, or indicates a heterogeneous protein sample (for example, due to degradation).

• •Always keep DNA stocks and IDR samples on ice and minimize freeze-thaw cycles (aliquot into smaller volumes). Snap-freeze aliquots of purified protein after using them.
• •Do not re-use left-over diluted stocks of protein (prepared in step-by-step method details, step 17C). Make a fresh dilution of working stock for each assay.
• •Minimize the time that the samples spend in the PCR block following the incubation reaction in step-by-step method details, step 19. Add the loading dye immediately and load the samples on the gel as quickly as possible.
• •Reduce the salt concentration in the 2× EMSA buffer to stabilize electrostatic interactions (adjust from the recipe in materials and equipment setup, 2× EMSA buffer).
• •Condense the time that the samples are sitting in the wells prior to electrophoresis (i.e., shorten step-by-step method details, step 29). Once the wells are sealed with LMP agarose, start the electrophoresis immediately.
• •Increase the agarose gel concentration by 1%–5% to help resolve DNA bands.
• •Due to their lack of structure, IDRs are prone to degradation. Verify that the purified protein has not partially degraded by performing SDS-PAGE gel electrophoresis followed by gel staining (for example, using Coomassie blue).

Problem 4

All DNA bands are streaked or smeared (see example in Figure 4C).

Potential solution

Smeared or streaked bands indicate irregular agarose gel polymerization or gel heating at several different steps in the step-by-step method details section summarized below.

• •While microwaving the 1% agarose gel in steps 13B-D, make sure that the agarose is sufficiently mixed into the TAE buffer. Swirl the flask vigorously in between each step and ensure that all agarose has dissolved before letting the flask cool and pouring the gel.
• •Ensure that you are only adding 16 mg of LMP agarose to 1 mL of 1× TAE in step 21A.
• •If not thoroughly mixed with the 1× TAE, LMP agarose tends to clump at the bottom of 1.5 mL Eppendorf tubes. Mix the LMP agarose aliquots well by vortexing before and after boiling at 95°C in steps 21B-D. Look out for agarose clumps at the bottom of the tube.
• •After the LMP agarose boiling step, let it fully cool to 33°C in the thermomixer before adding it to the binding reactions in step 26. We typically allow for at least 15 min of incubation at 33°C prior to use.
• •Band smearing could also be caused by excessive gel heating during electrophoresis in step 32. It is important to run the gel in a 4°C room to combat this issue.
• •If using 50× TAE prepared in-house for the agarose gel and running buffer, verify that the concentration of each component in the buffer is at 1× in the final solution. The 50× stock solution (recipe in materials and equipment setup, 50× TAE) must be diluted in filter sterilized MilliQ H₂O before use.

Problem 5

IDR-DNA complex bands migrate in irregular patterns (see example in Figure 4D).

Potential solution

This indicates that there may have been a pipetting or mixing error involving the protein of interest.

• •Mix the working protein dilution(s) made up in step-by-step method details, step 17C well before adding it to each binding reaction. Collect all liquid at the bottom of the tube by centrifugation.
• •Check that all calculations for the volume of diluted protein to be added to each binding reaction are correct (for example, those in Table 1).

Problem 6

Non-specific signals in the DNA gel interfere with DNA band quantification (see example in Figure 4E).

Potential solution

The presence of non-specific signals is likely due to contamination during the preparation of the agarose gel, TAE running buffer, or staining solution.

• •Always wear gloves and spray all surfaces with 70% ethanol.
• •Clean the gel tray and staining container thoroughly with 70% ethanol as outlined in step-by-step method details, step 13G-H and step 33A-B. Use Kimwipes and not paper towel to wipe the inside of the vessels. Preferably, air drying is best as it limits the chances of introducing fibers into the vessels.
• •Spray and clean the glass surfaces of the D-DiGit gel imager with 70% ethanol and rinse with filtered MilliQ H₂O to remove traces of nucleic acids from previous gels. Use Kimwipe sheets to clean.

Problem 7

DNA migration is unchanged in the presence of the IDR of interest (see example in Figure 4F).

Potential solution

Unless you expect that your protein should not bind DNA, this suggests that protein concentration may need to be adjusted.

• •Use a higher protein concentration range in the binding reactions than that suggested in step-by-step method details, step 17C.
• •If the IDR of interest is negatively charged, it may be co-migrating with the negatively charged DNA substrate. At least one report has shown that reducing the pH of the 2× EMSA buffer and 1× TAE running buffer to ∼6.0 helps to combat this.¹⁵
• •To ensure that the issue lies with the protein of interest and not with the gel or electrophoresis conditions, it is useful to include a known DNA-binding protein as a positive control (if this is available).

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Damien D’Amours (damien.damours@uottawa.ca).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Alyssa Pastic (apast053@uottawa.ca).

Materials availability

This study did not generate unique reagents.

Data and code availability

This study did not generate original code or analyze datasets.

Acknowledgments

This work was supported by a CIHR grant to D.D. (CIHR FDN–167265) and an OGS scholarship to A.P. Work in D.D.’s laboratory is supported by a Canada Research Chair in Chromatin Dynamics & Genome Architecture (CRC-2017-00064). The following vector images for the graphical abstract were obtained from Bioicon (https://bioicons.com/?query) under the following licenses: electrophoresis chamber icon, micropipette icon, and microtube icon by Servier (https://smart.servier.com/) is licensed under CC-BY 3.0 Unported https://creativecommons.org/licenses/by/3.0/; gel imager icon and purification column icon by DBCLS (https://togotv.dbcls.jp/en/pics.html) is licensed under CC-BY 4.0 Unported https://creativecommons.org/licenses/by/4.0/; and PCR_icon by KeHan (https://github.com/kehantan) is licensed under CC0 https://creativecommons.org/publicdomain/zero/1.0/. The gel staining container icon was obtained from Adobe stock images by Jhanto (https://stock.adobe.com/ca/contributor/211944857/jhanto?load_type=author&prev_url=detail).

Author contributions

Conceptualization, A.P., A.K., and D.D.; data curation, A.P. and A.K.; methodology, A.P., A.K., and D.D.; writing – original draft, A.P.; writing – review and editing, A.K. and D.D.; funding acquisition, D.D.; supervision, D.D.

Declaration of interests

The authors declare no competing interests.