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Protein and antibody purification followed by immunoprecipitation of MYB and GATA zinc finger-type maize proteins with magnetic beads

Summary Co-immunoprecipitation (Co-IP) is a widely used and powerful approach for studying protein-protein interactions in vivo. Here, we describe a protocol for antibody purification and immobilization followed by immunoprecipitation from plant tissue extracts using magnetic beads. The protocol has been used to detect regulators in the Zea mays phenylpropanoid pathway. The protocol is amenable to a variety of downstream assays, including western blotting and mass spectrometry. For complete details on the use and execution of this protocol, please refer to Vélez-Bermúdez et al. (2015).


MATERIALS AND EQUIPMENT
Note: We suggest using molecular biology grade reagents and to autoclave and filter all the following buffers.
Note: Adjust the pH to 5.8 using sodium hydroxide (NaOH). Autoclave for 15 min and add the ampicillin just before inoculation with 5 mL culture.
CRITICAL: Sodium hydroxide (NaOH) is a base and caustic substance. It is highly recommended to wear protecting gloves, clothing, eye, and face protection.
Note: Prepare immediately before use, cOmplete, EDTA-free protease inhibitor cocktail dilute to 13 and 1 M Maltose.
Note: Sterilize with a 0.22 mm filter. Note: Prepare immediately before use.
Note: Prepare immediately before use.
CRITICAL: Methanol is a wood alcohol highly flammable and toxic. Avoid contact with skin and eyes. Avoid inhalation of vapor and keep away from sources of ignition.
Note: For the affinity purification of antibodies, prepare PBS -Phosphate-buffered saline 13 + 0.1% TWEENâ 20 and a 0.2 M of glycine solution adjusted to pH 2.6 with HCl and sterilize with a 0.22 mm filter. The 5% bovine serum albumin solution should be freshly made and keeping at 4 C until use.
CRITICAL: Glycine is a proteinogenic amino acid that may cause irritation. Avoid contact with eyes, skin, and clothing.
CRITICAL: Hydrochloric acid also known as muriatic acid, is a corrosive substance and toxic if inhaled. Handle the reagent while under a fume hood, wear a chemical-resistant apron, gloves, and goggles.
CRITICAL: Phenylmethanesulfonyl fluoride is a serine protease inhibitor that can cause acute oral toxicity, skin, and eye irritation. It is recommended to wear protective gloves, googles, clothing, and face protection.

STEP-BY-STEP METHOD DETAILS
Protein expression and purification-3 days This first part of the protocol is an optimization of the pMALä Protein Fusion & Purification System protocol (NEB #E8200S). pMAL is one of the most used systems for the purification of fusion proteins. An advantage of this method is the fact that maltose-binding protein (MBP) binds to a positively charged resin at pH 5.5, where most other proteins do not bind. Moreover, the MBP-fusion protein can be purified in a one-step MBP affinity purification with high quality. This step is critical since the resultant purified protein will be used for the second step of this protocol.

Pre-culture
Timing: 10 min; day 1 Note: For this protocol we used the pDESTH1 vector (Ampicillin resistant).
1. Add 5 mL LB medium, 0.2% glucose, and 10 mL Ampicillin (stock 50 mg/ mL) to a culture tube. 2. Inoculate with a single bacterial colony from the agar plate (from day 0). 3. Culture at 37 C and shake for 16 h.
Note: The LB medium can be stored at 20 C-25 C for 1 month. 50% glucose can be stored at 4 C for 6 months and Ampicillin should be store at -20 for up to 6 months.

Protein induction
Timing: 5-6 h; day 2 4. In an Erlenmeyer flask, inoculate 500 mL of rich medium glucose (0.2%) and Ampicillin (100 mg/ mL) with 5 mL of a culture of cells grown for 16 h containing the fusion plasmid. Mix well and measure the starting OD of the culture.
Note: The rich medium can be stored at 20 C-25 C for up to 1 month. Note: Glucose is necessary in the growth medium to repress the maltose genes on the chromosome of the E. coli host, one of which is an amylase that can degrade the amylose on the affinity resin.
5. After 2 h, measure the optical density (OD) of the culture A 600 $ 0.5 (2 3 108 cells/mL), add IPTG to a final concentration of 1 mM, and incubate the culture at 37 C for 3 h.
Note: IPTG can be stored at À20 C in dark for up to 9 months.
Note: The time and temperature until the OD of the culture reach an optical density of 0.5 can vary with the host strain and the stability of the protein.
6. Divide the induced culture to two 500 mL polypropylene bottles (e.g., Beckman Coulter), centrifuge the culture at 4,000 3 g for 20 min at 4 C, discard the supernatant, resuspend the pellets in 25 mL column buffer (500 mL of culture), and freeze the sample for 16 h at À20 C.
CRITICAL: To avoid degradation of the protein, add 0.1 mM PMSF (phenyl methylsulfonyl fluoride) and protease inhibitor cocktail (13). Harvest the cells quickly and keep them chilled.
Note: PMSF can be stored at À20 C in dark; it should be stable for at least two years. The column buffer can be stored at 4 C for up to 6 months.
Pause point: The resuspended pellet can be stored at À20 C for up to one month.

Protein purification
Timing: 5-6 h; day 3 7. Thaw the sample in cold water. 8. Transfer the sample to a 50 mL Nalgene centrifuge tube, place in an ice bath, and sonicate in short pulses of 15 s.
Note: Keep the samples on ice to avoid degradation of the protein.
9. Centrifuge the sample at 20,000 3 g for 20 min at 4 C, keep the supernatant (crude extract), and dilute the crude extract 1:2 with column buffer (total = 50 mL).
Note: It is important to add 0.1 mM of PMSF (phenyl methylsulfonyl fluoride) and protease inhibitor cocktail (13) to avoid degradation of the protein.
Note: The binding capacity of the amylose resin is 2-3 mg fusion protein/mL bed volume.
11. Wash the column five times with 15 mL column buffer. 12. Load the diluted crude extract at a flow rate of no more than 1.87 mL/min.

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13. Wash the column with 36 mL of column buffer at a flow rate of no more than 3.75 mL/min.
14. Elute the fusion protein with column buffer + 10 mM maltose and collect 15 fractions of 500 mL each.
Note: The elution buffer (column buffer + 10 mM maltose) should be prepared fresh.
Note: The maltose solution can be stored at 4 C for 3 days; for long-term storage (6 months), stock solutions should be stored at À20 C. It is recommended that the maltose solution is prepared and used at the same day.
15. Run 15 mL of each elution in an acrylamide gel and quantify using Bradford protein assay, the fractions containing the purified protein (Figure 1). 16. If the concentration is lower than desired, pool the protein-containing fractions and concentrate to about 1 mg/mL using an Amicon Ultra-0.5 centrifugal filter unit.
Note: The pore size of the Amicon Ultra centrifugal filter should be selected according to the molecular weight of the purified protein.
17. Perform a western blot using 5 mg of purified protein to check the quality. A representative western blot is shown in Figure 2.
Pause point: The purified protein can be stored for 1 day on ice at 4 C.

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This second part of the protocol is critical for a successful immunoprecipitation assay. Currently, different protocols for affinity purification of antibodies are available. Here, we offer an inexpensive and accurate method to obtain a pure and intact antibody for the IP experiment.
18. Take 800 mg of the purified protein and run on an SDS-PAGE gel.
Note: We suggest using Nupage 4-12 Bis-Tris gel 1.5 mm 3 10 wells (Invitrogen, NP0335B0X) and a run time of 38 min. The sample can be loaded into the lanes 1-8. Keep two lanes empty and use the last lane for the protein marker ( Figure 3A).
Note: The BIO-RAD transfer buffer should be stored at 4 C before use for at least 30 min.
Note: This protocol can be use when the amount of purified protein is at least 230 mg.
19. Transfer the protein onto a membrane. 20. Stain the membrane with Ponceau S ( Figure 3B). 21. Locate the size corresponding to your protein, cut out the band, and mark the protein side (Figure 3C). 22. De-stain the strip with 13 PBS-Tween 0.1% and block with 5% BSA (fatty acid-free) in 10 mL 13 PBS for 1 h at 25 C.
Note: PBS 13 can be stored at 20 C-25 C for up to 1 year. BSA should be prepared immediately before use and stored at 4 C.
23. Wash (by shaking) the strip 5 times with 10 mL 13 PBS-Tween 0.1%. 24. Incubate the strip with the antibody serum for 16 h at 4 C.
Note: Since in our case the specific antibody constitutes 1%-10% of the total antibody pool, we have used at least 5 mL of the antibody (if the concentration of the antibody is $1 mg/mL) and 1 mg of the antigen (purified protein).
Note: 1 mg of purified protein can theoretically bind around 0.5-2 mg of the specific antibody.
Note: Since the antibodies have been produced in rabbit, this step is required to obtain a clean antibody for the immunoprecipitation.
25. Remove the serum. Note: We suggest storing the antibody at À20 C until validation of the purified antibody.
26. Wash the strip twice with 10 mL 13 PBS-Tween 0.1% during 5 min each time. 27. Wash the strip twice with 10 mL 13 PBS during 5 min each time.
Note: Measure the last wash with 13 PBS spectrophotometrically at A 280 to make sure that the optical density it is below 0.1.
28. Collect 11 elutions of the purified antibody bound to the strip with 0.2 M glycine pH 2.6 in 1.5 mL Eppendorf tubes.
Note: The glycine solution can be stored at 20 C-25 C for up to one month.
Note: 1 M Tris pH 8.0 buffer is used to neutralize the glycine.
Note: Wash the strip 3 times with 13 PBS for 5 min each time and store samples at À20 C for further use.
29. Store the elutions at À20 C until use.
Pause point: The purified antibody can be stored at À20 C for 1 month or at À80 C for up to 2 years. 30. Perform a western blot using recombinant protein and total plant protein extract to verify the quality of the different fractions of the purified antibody ( Figure 4).

Immunoprecipitation of protein complexes-2 days
Timing: 10 min; day 6 Immunoprecipitation is one of the most widely used methods for studying protein-protein interactions. Here, an antibody against a specific target protein is used to detect an immune complex with that target under a particular condition. This part of the protocol details the associated procedures of co-immunoprecipitation and considerations associated with this experiment. The use of a clean and specific secondary antibody is critical for the successful detection of a specific putative partner through western blot analysis. 32. Transfer 50 mL (1.5 mg) of Dynabeads protein A into a 2 mL low-binding protein Eppendorf tube for each immunoprecipitation (antibody), place on the magnet and discard the supernatant. 33. Wash the Dynabeads three times with 200 mL 13 PBS+1 mM PMSF.

Note:
The washing buffer for Dynabeads should be prepared immediately before use and stored at 4 C until use.
Note: During the IP protocol, the Dynabeads should be washed by turning the Eppendorf tubes 4 full turns without removing the tube from the magnet. Discard the supernatant each time, it is important to avoid that the Dynabeads dry out.
Note: We suggest using low-binding protein Eppendorf tubes for all the steps involving Dynabeads.
34. Incubate the Dynabeads with the purified antibodies for 16 h at 60 rpm rotation at 4 C.
Note: 50 mL of Dynabeads correspond to 1.5 Ab/mg beads for IgG.  Note: For an efficient immunoprecipitation the amount of purified antibody is typically between 5 to 7 mg/mL of Dynabeads.
Cell lysate and Co-IP Timing: 10 min; day 7 35. Grind 1 g plant material in liquid nitrogen in a pre-chilled mortal to a fine powder, resuspend in 500 mL extraction buffer A or B, and incubate in ice during 30 min with 5 min vortex every 5 min.
CRITICAL: The extraction buffer (Option A) is supplied in the Dynabeadsä Co-Immunoprecipitation Kit (Invitrogen). In case of selecting this buffer, continue the process using the buffers included in the kit.
Note: The extraction buffer (Option A) should be prepared immediately before use and stored at 4 C. IP buffer (Invitrogen Co-IP kit, 103) can be stored at 20 C-25 C according to the manufacturer's instructions. NaCl can be stored at 20 C-25 C for up to 28 days. DL-dithiothreitol (DTT) can be stored at 4 C for 1 year or indefinitely at À20 C in dark conditions. MG132 can be stored at À20 C for up to 1 year. cOmplete, EDTA-free protease inhibitor cocktail tablets should be stored dry at 4 C; stock solutions can be stored at 4 C for 1-2 weeks or at À20 C for at least 12 weeks. Phenylmethylsulfonyl fluoride can be stored at À20 C for at least two years.
Note: The extraction buffer B is described in Abraham-Juá rez (2019). Buffer B is suitable for proteins that are dynamically transported between nucleus and cytosol in native conditions, and can be used as an alternative to the kit. If your protein has another subcellular localization or some specific conditions, the buffer should be optimized for this purpose.
Note: The extraction buffer (Option B) should be prepared immediately before use and stored at 4 C. Tris-HCl, pH 7.4 (Stock 1 M) may be stored at 2 C-8 C for at least 1 year. NaCl can be stored at 20 C-25 C for up to 28 days. IGEPAL-CA-630 can be stored at 8 C-25 C in a cool and dry area for up to 5 years. MG132 can be stored at À20 C for up to 1 year.
CRITICAL: Liquid nitrogen is a cryogenic that expands 695 times in volume when it vaporizes, has no warning properties such as odor or color, and may cause asphyxiation hazards. Use protective clothing and handle in well-ventilated areas.
36. Centrifuge at 11,200 3 g at 4 C for 10 min. 37. Collect the supernatant and transfer to a fresh Eppendorf tube. 38. Quantify the total protein from the lysate using a protein assay.
Note: We use the Pierce 660 nm protein assay (Thermo Fisher).
39. Wash twice the Dynabeads-Ab complex with 13 PBS + 1 mM of PMSF during 5 min each time at 4 C. 40. Incubate the Dynabeads-Ab complex with 500 mL (1.5 mg) of the lysate for 3 h at 4 C in rotation.
Keep 100 mL of the lysate for the input.
Note: Depending on the stability and abundance of the protein complex of interest, you can use 1-20 mg of total protein extract.
Note: The incubation time for the Dynabeads-Ab complex + lysate depends on the antibody. For purified antibodies, we suggest to incubate 3 h, commercial antibodies should be incubated for 1 h to avoid background.
41. Wash the Dynabeads-Ab complex three times with 200 mL washing buffer 1 for 5 min each time at 4 C. 42. Wash the Dynabeads-Ab-Ag complex once with 200 mL washing buffer 2 in rotation at 30 rpm for 5 min at 4 C.
Note: Washing buffers 1 and 2 should be prepared immediately before use and stored at 4 C.
43. Place the Dynabeads-Ab-Ag complex on the magnet and discard the supernatant, immediately resuspend in 35 mL 1.53 sample buffer, and boil at 95 C for 5 min.
Note: 1.53 Sample buffer can be stored at 4 C for at least 1 year. Tris-HCl, pH 6.8 (Stock 1 M) can be stored at 2 C-8 C for at least 1 year. 2-mercaptoethanol (14.3 M) can be stored at 2 C-8 C for at least 3 years. Glycerol 50% can be stored at À20 C for 2 weeks or indefinitely at À80 C.
Store stock solution of sodium dodecyl sulfate solution (10% w/v) at 25 C for use with a recommended minimum shelf life of 3 years. Bromophenol Blue sodium salt can be stored at 20 C-25 C for up to 2 years.
44. Place the Dynabeads-Ab-Ag complex on the magnet and recover the supernatant in a fresh Eppendorf tube.
Note: You can collect a second elution of 30 mL and recover in another Eppendorf tube (do not mix elutions).
Pause point: The samples can be stored at À80 C until immunoblotting or mass spectrometry analysis.
Note: Keep 100 mL of lysis sample to use as a control for the experiment.
Note: To avoid background during western blot analysis, we suggest using Clean-Blotä IP detection reagent (HRP), Thermo Scientific, cat# 21230 for polyclonal antibodies.
Note: For mass spectrometry analysis, you can also make the digestion directly in the beads.

EXPECTED OUTCOMES
Co-IP is a useful tool to demonstrate protein-protein interactions and interactions between protein complexes. Following this detailed protocol, we were able to identify a novel set of putative interaction partners in Arabidopsis through Co-IP followed by sequencing, and successfully confirmed direct interaction between two proteins in maize using Co-IP experiments followed by western blot analysis. It is highly recommended that the experiments are performed multiple times to standardize the conditions to study the proteins of interest.

LIMITATIONS
The main and critical limitation for the Co-IP experiment is the quality of the antibody. We have included here our method for purifying antibodies, which has been successfully employed to purify various custom-made antibodies raised against proteins in maize and Arabidopsis. For Co-IP experiments preceding sequencing, it is critical to standardize the experiment to avoid non-specific binding to the Dynabeads or a low amount or stringency in the washes, which may lead to false positives in the first and false negatives in the second case. In Co-IP experiments that are followed by western blot analysis, it is very important to use highly specific primary and secondary antibodies, since lack of specificity may result in unwanted background signal. If this is the case, we suggest using Clean-Blotä IP detection reagent (HRP) as a secondary antibody for polyclonal antibodies to avoid the interference with both heavy and light chains of IgG, which can cause problems to resolve western blot with proteins with a molecular weight of 50 or 25 kDa. For mass spectrometry analysis, we suggest to use the transgenic plant expressing the empty vector as a negative control. If an antibody against the endogenous protein is used, Dynabeads without antibody, or the pre-immune bleed can be used as a negative control.

Problem 1
Low amount or degradation of the purified protein (steps 15 and 16). The presence of ZML2 and MYB11 was detected by immunoblot analyses using ZML2 and MYB11 antibodies. Input extracts from 9-d-old maize leaves were immunoprecipitated with MYB11 antibody, and ZML2 was detected by immunoblot. The presence of MYB11 in the immunoprecipitated samples was detected as a control. Adapted from Vé lez-Bermú dez et al. (2015).

Problem 5
High background and nonspecific bands in the Co-IP western blot (step 45).

Potential solutions
Use less antibody to prevent binding of non-specific proteins to the beads.
Reduce the amount of sample loaded onto the Dynabeads to prevent non-specific binding of proteins to the antibody.
Reduce the incubation time samples/Dynabeads.
To avoid degradation of the antibody during the Co-IP, add fresh protease inhibitors and MG132.
Use the Clean-Blotä IP detection reagent (HRP) during developing the western blot.

Problem 6
High amount of antibody eluting with the target protein (step 45).

Potential solutions
Crosslink the antibody with the Dynabeads before running the Co-IP experiment (step 45).

Potential solutions
Increase the amount of antibody.
Ensure to elute the target proteins from the Dynabeads.
Do not add non-ionic detergents such as NP-40 or Triton X-100 to the lysis buffer.
Use less stringent washing buffer conditions and reduce the number of washing steps.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Wolfgang Schmidt (wosh@gate.sinica.edu.tw).

Materials availability
Unique reagents (i.e., plasmids or purify antibodies) generated in this study can be requested from Wolfgang Schmidt (wosh@gate.sinica.edu.tw).

Data and code availability
The published article includes the figures generated with this protocol.

ACKNOWLEDGMENTS
We thank Erich Grotewold (Biochemistry and Molecular Biology, Michigan State University) for supporting the production of ZmMYB11 antibody. David Caparró s-Ruiz was supported by the Spanish project AGL2014-58126-R funded by the Ministerio de Economía y Competitividad /AEI and European Regional Development Fund (FEDER). This work was also supported by the SGR programs (SGR2009-GRC703 and 2017SGR710) from the Secretaria d'Universitats i Recerca del Departament d'Empresa i Coneixement de la Generalitat de Catalunya and by the CERCA Programme/Generalitat de Catalunya. We also acknowledge financial support from ''Severo Ochoa Programme for ll OPEN ACCESS