ll OPEN ACCESS Protocol Protein and antibody purification followed by immunoprecipitation of MYB and GATA zinc finger-type maize proteins with magnetic beads Isabel Cristina Vélez- Bermúdez, Jorge Enrique Salazar- Henao, Marta Riera, David Caparros- Ruiz, Wolfgang Schmidt icvb@gate.sinica.edu.tw (I.C.V.-B.) wosh@gate.sinica.edu.tw (W.S.) Highlights Protein expression and purification of MBP fusions Affinity purification of antibodies Immunoprecipitation from maize cell extracts with magnetic beads Amenable to Co-immunoprecipitation (Co-IP) is a widely used and powerful approach for studying protein- downstream assays protein interactions in vivo. Here, we describe a protocol for antibody purification and like mass spec and immobilization followed by immunoprecipitation from plant tissue extracts using magnetic western blot 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. Vélez-Bermúdez et al., STAR Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional Protocols 3, 101449 guidelines for laboratory safety and ethics. June 17, 2022 ª 2022 The Author(s). https://doi.org/10.1016/ j.xpro.2022.101449 ll OPEN ACCESS Protocol Protein and antibody purification followed by immunoprecipitation of MYB and GATA zinc finger-type maize proteins with magnetic beads Isabel Cristina Vélez-Bermúdez,1,7,8,* Jorge Enrique Salazar-Henao,1,7 Marta Riera,6 David Caparros-Ruiz,6 and Wolfgang Schmidt1,2,3,4,5,9,* 1Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan 2Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica and National Chung-Hsing University, Taipei 11529, Taiwan 3Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 40227, Taiwan 4Biotechnology Center, National Chung-Hsing University, Taichung 40227, Taiwan 5Genome and Systems Biology Degree Program, College of Life Science, National Taiwan University, Taipei 10617, Taiwan 6Centre de Recerca en Agrigenòmica, Consortium CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, 08193 Barcelona, Spain 7These authors contributed equally 8Technical contact 9Lead contact *Correspondence: icvb@gate.sinica.edu.tw (I.C.V.-B.), wosh@gate.sinica.edu.tw (W.S.) https://doi.org/10.1016/j.xpro.2022.101449 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). BEFORE YOU BEGIN Reagent preparation Timing: 2–4 h Note: This protocol has been successfully used for transiently expressed proteins in tobacco and Arabidopsis. Here, we describe the specific steps for maize. The day before the start of the experiment (day 0): 1. Prepare LB, 50% glucose, column buffer, the rich broth, and autoclave. Note: The glucose should not be autoclaved for more than 15 min to avoid degradation and release of toxic substances. CRITICAL: We used the pMAL purification system (NEB #E8200S). STAR Protocols 3, 101449, June 17, 2022 ª 2022 The Author(s). 1 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ll OPEN ACCESS Protocol Note: Inoculate 500 mL of rich broth for the purification of each protein. 2. Prepare stocks of antibiotics and IPTG. Note: For this protocol we prepared a stock of 50 mg/mL of Ampicillin, the antibiotics can vary according with the vectors used. The IPTG should be prepared to a stock of 0.1 M. Both re- agents can be sterilized with a 0.22 mm filter. Note: The antibiotics depend on the selection of the plasmid-encode genes used for the pro- tein expression. 3. Streak a single colony of the bacteria carrying the plasmid in a LB + antibiotic plate and incubate for 18 h at 37 C. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-ZmMYB11. Working dilution: (Vélez-Bermúdez et al., 2015) https://academic.oup.com/plcell/ 1:100 article/27/11/3245/6096690?login=false Rabbit polyclonal anti-ZmZML2. Working dilution: (Vélez-Bermúdez et al., 2015) https://academic.oup.com/plcell/ 1:100 article/27/11/3245/6096690?login=false ECL Rabbit IgG, HRP-linked whole Ab from donkey GE Healthcare NA934 Bacterial and virus strains ECOS 21 Competent Cells Yeastern Biotech Cat#FYE207-40VL Rosetta(DE3) Competent Cells Novagen Cat#70954 Biological samples Maize (Zea mays) B73 leaves (9 days old) USDA https://npgsweb.ars-grin.gov/ gringlobal/search Chemicals, peptides, and recombinant proteins BD Difco Dehydrated Culture Media: LB Broth, Miller BD Difco Cat#BD 244620 (Luria-Bertani) BD Difco Dehydrated Culture Media: LB Agar, Miller BD Difco Cat#BD 244520 (Luria-Bertani) D-(+)-Glucose Sigma-Aldrich Cat#G8270 Bacto Tryptone Thermo Fisher Scientific Cat#211669 Yeast Extract Merck Cat#70161 Sodium chloride (NaCl) Merck Cat#7647-14-5 Ampicillin Sigma-Aldrich Cat# A9393 Phenylmethylsulfonyl fluoride (PMSF) Merck Cat#11359061001 Isopropyl b-D-thiogalactoside, Isopropyl b-D-1- Merck Cat#I6758 thiogalactopyranoside (IPTG) cOmplete, EDTA-free protease inhibitor cocktail (1 Roche Cat# 11873580001 unit/253) Bradford Reagent Merck Cat#B6916-500ML Amylose Resin Biolabs Cat#E8021S D-(+)-Maltose monohydrate Sigma-Aldrich Cat# M9171 Trizma hydrochloride solution (pH 7.4) Sigma-Aldrich Cat#T2194-1L UltraPure 1 M Tris-HCI, pH 8.0 Invitrogen Cat#15568025 Tris-HCl (pH 6.8) Abcam Cat#ab286853 UltraPure 0.5 M EDTA, pH 8.0 Invitrogen Cat#15575020 Nupage 4–12 Bis-Tris Gel Thermo Fisher Scientific Cat#NP0335B0X Ponceau S Solution Abcam Cat#ab270042 Bovine Serum Albumin (BSA) Sigma-Aldrich Cat# A2153 PBS - Phosphate-Buffered Saline (103) pH 7.4, RNase- Invitrogen Cat# AM9625 free (Continued on next page) 2 STAR Protocols 3, 101449, June 17, 2022 ll Protocol OPEN ACCESS Continued REAGENT or RESOURCE SOURCE IDENTIFIER TWEEN 20 Sigma-Aldrich Cat#P1379 Glycine Sigma-Aldrich Cat#G7126 Hydrochloric acid Sigma-Aldrich Cat#320331 Sodium hydroxide solution Sigma-Aldrich Cat#2105 Dynabeads Protein A Invitrogen Cat#10001D MG-132, 10 mM (1 mL in DMSO) Selleckchem Cat#S2619 DL-Dithiothreitol solution Sigma-Aldrich Cat#43816 IGEPAL CA-630 Sigma-Aldrich Cat#I8896 2-Mercaptoethanol Sigma-Aldrich Cat#M6250 Glycerol Sigma-Aldrich Cat#G5516 Sodium dodecyl sulfate solution, Molecular Biology Promega Cat#V6551 Grade (10% w/v) Bromophenol Blue sodium salt Sigma-Aldrich Cat#B5525 PageRuler Prestained Protein Ladder, 10–180 kDa Thermo Fisher Scientific Cat#26616 NuPAGE MES SDS Running Buffer (203) Thermo Fisher Scientific Cat# NP0002 Methanol Merck Cat#106009 Autoclaved distilled water n/a N/A Liquid nitrogen n/a N/A Recombinant ZmMYB11-MBP (Vélez-Bermúdez et al., 2015) https://academic.oup.com/plcell/ article/27/11/3245/6096690?login=false Recombinant ZmZML2-MBP (Vélez-Bermúdez et al., 2015) https://academic.oup.com/plcell/ article/27/11/3245/6096690?login=false Critical commercial assays pMAL Protein Fusion & Purification System New England Biolabs Cat#E8200S Trans-Blot Turbo RTA Mini 0.2 mm PVDF Transfer Kit, Bio-Rad Cat#170-4272 for 40 blots Dynabeads Co-Immunoprecipitation Kit Invitrogen Cat#14321D Deposited data Raw and analyzed data (Vélez-Bermúdez et al., 2015) https://academic.oup.com/plcell/ article/27/11/3245/6096690?login=false Recombinant DNA Plasmid: pDONR207 Invitrogen https://www.snapgene.com/resources/ plasmid-files/?set=gateway_cloning_ vectors&plasmid=pDONR207 Plasmid: pDESTTH1 (Hammarström et al., 2002) https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC2373440/ Full-length cDNA encoding ZmMYB11 (Vélez-Bermúdez et al., 2015) https://academic.oup.com/plcell/ article/27/11/3245/6096690?login=false Full-length cDNA encoding ZmZML2 (Vélez-Bermúdez et al., 2015) https://academic.oup.com/plcell/ article/27/11/3245/6096690?login=false Other Safe lock tubes 2.0 mL Eppendorf Cat#30120094 Eppendorf LoBind microcentrifuge tubes Merck Cat#EP0030108302-100EA Amicon Ultra-4 centrifugal filter unit Millipore Cat#UFC805024 Pipette filter tips, 10 mL Labcon Cat# 1051-800-000-9 Pipette filter tips, 200 mL Labcon Cat# 1059-800-000-9 Pipette filter tips, 1000 mL Labcon Cat# 1097-965-008-9 Eppendorf centrifuge Eppendorf Cat#5417R Trans-Blot Turbo Transfer System Bio-Rad Cat#1704150 Econo-Pac chromatography columns Bio-Rad Cat#732-1010 Sonicator Ultrasonic Processor Misonix, Inc. Cat# S-4000 PowerWave XS2, microplate spectrophotometer Agilent Technologies N/A MF-Millipore membrane filter, pore size 0.22 mm Millipore Cat#GSWP04700 Mini gel tank Invitrogen Cat#A25977 BioVoltTM power supply BioProducts Cat#SBE 250 Cold room N/A N/A (Continued on next page) STAR Protocols 3, 101449, June 17, 2022 3 ll OPEN ACCESS Protocol Continued REAGENT or RESOURCE SOURCE IDENTIFIER Mini heating dry bath incubator, MD-MINI Ms major science N/A DynaMag-2 magnet Invitrogen Cat#12321D Vortex-Genie 2 Scientific Industries, Inc. Cat#SI-0236 ELMI Intelli-Mixer RM-2L ELMI N/A MATERIALS AND EQUIPMENT Note: We suggest using molecular biology grade reagents and to autoclave and filter all the following buffers. Rich medium, glucose and ampicillin Reagent Final concentration Amount Tryptone 1% 10 g Yeast extract 0.5% 5g NaCl 0.5% 5g Glucose 0.2% 2g ddH2O N/A 999 mL Ampicillin (Stock 100 mg/mL) 100 mg/mL 1 mL Total N/A 1L 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 recom- mended 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. Column buffer Reagent Final concentration Amount Tris-HCl, pH 7.4 (Stock 1 M) 20 mM 20 mL NaCl 200 mM 11.7 g EDTA (Stock 0.5 M) 1 mM 2 mL ddH2O sterile N/A Up to 1 L Total N/A 1L Note: Sterilize with a 0.22 mm filter. Elution buffer Reagent Final concentration Amount Maltose (Stock 1 M) 10 mM 1 mL Column buffer N/A 99 mL Total N/A 100 mL 4 STAR Protocols 3, 101449, June 17, 2022 ll Protocol OPEN ACCESS Note: Prepare immediately before use. NuPAGE MES SDS running buffer Reagent Final concentration Amount NuPAGE MES SDS running buffer (203) 13 50 mL ddH2O sterile N/A 950 mL Total N/A 1L Note: Prepare immediately before use. Transfer buffer BIO-RAD Reagent Final concentration Amount Transfer buffer BIO-RAD (53) 13 100 mL Methanol N/A 100 mL ddH2O sterile N/A 300 mL Total N/A 500 mL 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% TWEEN 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. Washing buffer Dynabeads Reagent Final concentration Amount PBS - Phosphate-buffered saline (103) 0.2 M 10 mL Phenylmethylsulfonyl fluoride (PMSF, Stock 100 mM) 1 mM 1 mL ddH2O sterile N/A Up to 89 mL Total N/A 100 mL 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. STAR Protocols 3, 101449, June 17, 2022 5 ll OPEN ACCESS Protocol NaCl Solution Reagent Final concentration Amount NaCl 5M 5.85 g ddH2O sterile N/A Up to 100 mL Total N/A 100 mL Note: Sterilize with a 0.22 mm filter. Extraction buffer (Option A) Reagent Final concentration Amount IP buffer (Invitrogen Co-IP kit, 103) 13 10 mL NaCl 100 mM 0.2925 g DL-dithiothreitol (Stock 1 M) 2 mM 0.1 mL MG132 (10 mM) 50 mM 0.25 mL cOmplete, EDTA-free protease inhibitor cocktail 23 2 tablets Phenylmethylsulfonyl fluoride (PMSF, Stock 100 mM) 1 mM 0.5 mL ddH2O sterile N/A 39.15 mL Total N/A 50 mL CRITICAL: DL-dithiothreitol (DTT) is a reducing agent that can cause skin, eye and respi- ratory irritation. It is advised to wear appropriate protective equipment. Extraction buffer (Option B) Reagent Final concentration Amount Tris-HCl, pH 7.4 (Stock 1 M) 50 mM 2.5 mL NaCl (Stock 5 M) 150 mM 1.5 mL IGEPAL-CA-630 0.75% 0.375 mL MG132 (10 mM) 50 mM 0.25 mL cOmplete, EDTA-free protease inhibitor cocktail 23 2 tablets ddH2O sterile N/A 45.375 mL Total N/A 50 mL Washing buffer 1 Reagent Final concentration Amount Tris-HCl, pH 7.4 (Stock 1 M) 50 mM 2.5 mL NaCl (Stock 5 M) 150 mM 1.5 mL IGEPAL-CA-630 0.25% 0.125 mL MG132 (10 mM) 50 mM 0.25 mL cOmplete, EDTA-free protease inhibitor cocktail 23 2 tablets ddH2O sterile N/A 43.125 mL Total N/A 50 mL Washing buffer 2 Reagent Final concentration Amount Tris-HCl, pH 7.4 (Stock 1 M) 20 mM 1 mL ddH2O sterile N/A 49 mL Total N/A 50 mL 6 STAR Protocols 3, 101449, June 17, 2022 ll Protocol OPEN ACCESS 1.53 Sample buffer Reagent Final concentration Amount Tris-HCl, pH 6.8 (Stock 1 M) 62.5 mM 3.125 mL 2-Mercaptoethanol (14.3 M) 2 mM 0.00699 mL Glycerol 10% 5 mL Sodium dodecyl sulfate solution, molecular biology 2% 10 mL grade (10% w/v) Bromophenol Blue sodium salt 0.01% 0.005 g ddH2O sterile N/A 31.868 mL Total N/A 50 mL CRITICAL: 2-Mercaptoethanol is very toxic; wear protective gloves, clothing, eye, and face protection, and manipulate the reagent as well the buffer under a chemical hood. CRITICAL: Sodium dodecyl sulfate (SDS) is an organic compound that can cause skin, eye, and respiratory irritation. Avoid inhalation of dusts, substance contact, and keep away from heat and sources of ignition. STEP-BY-STEP METHOD DETAILS Protein expression and purification—3 days This first part of the protocol is an optimization of the pMAL Protein Fusion & Purification System protocol (NEB #E8200S). pMAL is one of the most used systems for the purification of fusion pro- teins. An advantage of this method is the fact that maltose-binding protein (MBP) binds to a posi- tively 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 mea- sure the starting OD of the culture. Note: The rich medium can be stored at 20 C–25 C for up to 1 month. STAR Protocols 3, 101449, June 17, 2022 7 ll OPEN ACCESS Protocol Note: Glucose is necessary in the growth medium to repress the maltose genes on the chro- mosome 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 A600  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 20 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), centri- fuge 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 20 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 20 C in dark; it should be stable for at least two years. The col- umn buffer can be stored at 4 C for up to 6 months. Pause point: The resuspended pellet can be stored at 20 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: The sonication program parameters used were: amplitude: 37, process time: 2:00 min, pulse on: 0:15 s, pulse off: 0:15 s, temperature monitoring and shutdown: 115 C. 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 in- hibitor cocktail (13) to avoid degradation of the protein. 10. Transfer 3 mL of amylose resin to a 1.5 3 12 cm polypropylene column. 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. Note: The flow rate should be 1.87 mL/minute for a 1.5 cm column ([50 3 (1.5)2] mL/hour). 8 STAR Protocols 3, 101449, June 17, 2022 ll Protocol OPEN ACCESS Figure 1. Recombinant CPC-MBP fusion protein purification The figure shows an example of protein purification using the protocol described here. Fractions were analyzed by SDS–PAGE (MW molecular weight markers). E; elution fractions. 13. Wash the column with 36 mL of column buffer at a flow rate of no more than 3.75 mL/min. Note: The flow rate should be 3.75 mL/minute for a 1.5 cm column ([100 3 (1.5)2] mL/hour). 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 20 C. It is recommended that the maltose solution is pre- pared 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 west- ern blot is shown in Figure 2. Pause point: The purified protein can be stored for 1 day on ice at 4 C. Affinity purification of antibodies—2 days Timing: 10 min; day 4 STAR Protocols 3, 101449, June 17, 2022 9 ll OPEN ACCESS Protocol Figure 2. Immuno-detection of purified ZML2, ZML1-MBP, ZML2-MBP, and MBP proteins using purified antibodies raised against MBP To validate the quality of the purified protein, three different dilutions (50, 40 and 30 ng) of two MBP fusion proteins (ZML1-MBP and ZML2-MBP) and MBP alone as a control were loaded in a gel and a western blot was performed using antibodies raised against MBP. The red arrows indicate the presence of the proteins. Adapted from Vélez-Bermúdez et al. (2015). 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: This protocol is an optimization of the protocol provided by Agrisera (https://www.agrisera. com/en/info/affinity-purification-of-antibodies-low-amount-of-antigen-available.html). 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. 10 STAR Protocols 3, 101449, June 17, 2022 ll Protocol OPEN ACCESS Figure 3. Representative example of affinity antibody purification (A) Scheme of Bis-Tris gel showing how to load the protein; M indicates the line for the protein marker. (B) Ponceau S staining of the membrane after transferring the protein from the gel to visualize the size of the corresponding antigen. Purified protein is indicated by an arrow. (C) Membrane after removing the strip containing the protein of interest. The absence of the strip containing the purified protein is indicated by an arrow. Note: We use the Bio-Rad Trans-Blot Turbo Transfer System. Standard SD protocol, 30 min, and up to 1.0 A; 25 V. 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 (Fig- ure 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 immedi- ately 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. STAR Protocols 3, 101449, June 17, 2022 11 ll OPEN ACCESS Protocol Note: We suggest storing the antibody at 20 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 A280 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. Elution 0.2 M glycine pH 2.6 1 M Tris pH 8.0 Incubation time (minutes) E1 200 mL 60 mL 15 E2 320 mL 96 mL 30 E3 200 mL 60 mL 10 E4 200 mL 60 mL 10 E5 290 mL 87 mL 10 E6 385 mL 115.5 mL 10 E7 400 mL 120 mL 10 E8 405 mL 121.5 mL 10 E9 1270 mL 381 mL 10 E10 990 mL 297 mL 10 E11 1000 mL 300 mL 10 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 20 C for further use. 29. Store the elutions at 20 C until use. Pause point: The purified antibody can be stored at 20 C for 1 month or at 80 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 interac- tions. 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. 12 STAR Protocols 3, 101449, June 17, 2022 ll Protocol OPEN ACCESS Figure 4. Immuno-detection of ZmZML2 using the purified antibody raised against this protein (Left) Immunoblot using three different dilutions of the purified recombinant proteins ZML1-MBP, ZML2-MBP, and MBP (50, 40 and 30 ng). The membrane was hybridized with an antibody raised against ZmZML2, and a band of 80 KDa corresponding to the ZML2-MBP was detected. (Right) Western blot analysis using 50 mg of total protein extract from leaves of 9-day-old maize B73 plants hybridized with the purified antibody raised against ZmZML2. The native ZML2 is detected at 28 kDa. Adapted from Vélez-Bermúdez et al. (2015). Antibody coupling 31. Resuspend the Dynabeads protein A in a vial. Note: Do not vortex the Dynabeads, mix gently by hand. 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: The final volume (antibody + 13 PBS + 1 mM PMSF) should be 200 mL per 50 mL of Dynabeads. Note: 50 mL of Dynabeads correspond to 1.5 Ab/mg beads for IgG. STAR Protocols 3, 101449, June 17, 2022 13 ll OPEN ACCESS Protocol Note: For an efficient immunoprecipitation the amount of purified antibody is typically be- tween 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 Dynabeads Co-Immuno- precipitation 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 manufac- turer’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 20 C in dark conditions. MG132 can be stored at 20 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 20 C for at least 12 weeks. Phenylmethylsulfonyl fluoride can be stored at 20 C for at least two years. Note: The extraction buffer B is described in Abraham-Juárez (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 20 C for up to 1 year. CRITICAL: Liquid nitrogen is a cryogenic that expands 695 times in volume when it vapor- izes, 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 incu- bated for 1 h to avoid background. 14 STAR Protocols 3, 101449, June 17, 2022 ll Protocol OPEN ACCESS 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 20 C for 2 weeks or indefinitely at 80 C. Store stock solution of sodium dodecyl sulfate solution (10% w/v) at 25 C for use with a recommen- ded 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 Ep- pendorf tube. Note: You can collect a second elution of 30 mL and recover in another Eppendorf tube (do not mix elutions). 45. Use the first elution (35 mL) for western blot or mass spectrometry analysis (LC-MS/MS) (Figure 5). Pause point: The samples can be stored at 80 C until immunoblotting or mass spectrom- etry 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-Blot 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 inter- action 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 stan- dardize 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 exper- iments preceding sequencing, it is critical to standardize the experiment to avoid non-specific bind- ing 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 STAR Protocols 3, 101449, June 17, 2022 15 ll OPEN ACCESS Protocol Figure 5. Co-immuno-precipitation of ZmMYB11 and ZmZML2 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 Vélez-Bermúdez et al. (2015). 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- Blot 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 sug- gest 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. TROUBLESHOOTING Problem 1 Low amount or degradation of the purified protein (steps 15 and 16). 16 STAR Protocols 3, 101449, June 17, 2022 ll Protocol OPEN ACCESS Potential solutions Try to use another host strain such as E. coli Rosetta (DE3). Add glucose to the growth media to avoid degradation of the amylose on the affinity resin. Optimize the time and the temperature during expression depending on the stability of the protein and host strain. Harvest cells quickly and keep them chilled. Add proteinase inhibitors and PMSF. Avoid that the column is running dry. Problem 2 Poor quality of the purified protein (step 17). Potential solutions Avoid loading the column for more than 16 h, increase the number of washing steps, and wash the column at a maximum of 10 mL/min for a 2.5 cm column. Problem 3 Low concentration and quality of the purified antibody (step 30). Potential solutions Use at least 222 mg of purified protein. Use at least 10–15 mL of serum or IgY sample. Adjust the elution conditions and time for each specific antibody. Do not use glycine older than 1 month, since the pH may vary. Neutralize the glycine with 1 M Tris pH 8.0. It is also possible to concentrate the antibody using an Amicon Ultra centrifugal filter or a similar device. Problem 4 Weak or no signal in the Co-IP western blot (step 45). Potential solutions This problem may be caused by degradation of the proteins during either sampling or grinding. To prevent degradation, the tissue should be stored immediately at 80 C. To solve problems caused by grinding, avoid thawing the tissue before adding the extraction buffer. Low concentration of proteins obtained from Arabidopsis or maize during lysis. To solve this prob- lem, keep the samples and buffers at 4 C and increase the amount of protease inhibitors or MG132. We also suggest using 1 g of starting material and an amount 1:2 (w/v) of buffer. Low affinity or concentration of the antibody. To solve this problem, optimize the required antibody. Perform the Co-IPs in a cold room or at 4 C. STAR Protocols 3, 101449, June 17, 2022 17 ll OPEN ACCESS Protocol 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 pro- teins 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-Blot 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). Problem 7 No eluted target protein detected (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 ful- filled 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 sup- porting the production of ZmMYB11 antibody. David Caparrós-Ruiz was supported by the Spanish project AGL2014-58126-R funded by the Ministerio de Economı́a y Competitividad /AEI and Euro- pean 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/General- itat de Catalunya. We also acknowledge financial support from ‘‘Severo Ochoa Programme for 18 STAR Protocols 3, 101449, June 17, 2022 ll Protocol OPEN ACCESS Centres of Excellence in R&D’’ SEV-2015-0533 and CEX2019-000902-S both funded by MCIN/AEI/ 10.13039/501100011033. I.C.V.-B. was supported by a Ministry of Science and Technology Fellowship (104-2811-12-001-025) and Academia Sinica Postdoctoral Fellowship (L004). J.E.S.-H. was supported by Academia Sinica Postdoctoral Fellowship (L004). Work in the Schmidt laboratory is supported by Academia Sinica and the Ministry of Science and Technology, Taiwan. AUTHOR CONTRIBUTIONS I.C.V.-B. and J.E.S.-H. performed the experiments and optimized the protocols that are described in this method article. I.C.V.-B., J.E.S.-H., M.R., D.C.-R., and W.S. performed data analysis and wrote the protocol. W.S. provided funding and edited the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. REFERENCES Abraham-Juárez, M.J. (2019). Western blot in produced as fusion proteins in Escherichia coli. Grotewold, E., Gray, J., Solano, R., Schmidt, W., maize. Bio Protoc. 101, e3257. Protein Sci. 11, 313–321. https://doi.org/10.1110/ Pagés, M., et al. (2015). A MYB/ZML complex reg- ps.22102. ulates wound-induced lignin genes in maize. Plant Hammarström, M., Hellgren, N., van Den Berg, S., Cell 27, 3245–3259. https://doi.org/10.1105/tpc.15. Berglund, H., and Härd, T. (2002). Rapid screening Vélez-Bermúdez, I.C., Salazar-Henao, J.E., Fornalé, 00545. for improved solubility of small human proteins S., López-Vidriero, I., Franco-Zorrilla, J.M., STAR Protocols 3, 101449, June 17, 2022 19