Input components from 9-d-old maize leaves were immunoprecipitated with MYB11 antibody, and ZML2 was detected by immunoblot
Input components from 9-d-old maize leaves were immunoprecipitated with MYB11 antibody, and ZML2 was detected by immunoblot. ? Amenable to downstream assays like mass spec and western blot Publishers notice: Starting any experimental protocol requires adherence to local institutional guidelines for laboratory security and ethics. Mouse monoclonal to TDT Co-immunoprecipitation (Co-IP) is usually a widely used and powerful approach for studying protein-protein interactions phenylpropanoid pathway. The protocol is usually amenable to a variety of downstream assays, including western blotting and mass spectrometry. Before you begin Reagent preparation Timing: 2C4 h This protocol has been successfully utilized for transiently expressed proteins in tobacco and Arabidopsis. Here, we describe the specific actions for maize. The day before the start of the experiment (day 0): 1. Prepare LB, 50% glucose, column buffer, the rich broth, and autoclave. 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). Inoculate 500?mL of rich broth for the purification of each protein. 2. Sulfacarbamide Prepare stocks of antibiotics and IPTG. 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 reagents can be sterilized with a 0.22?m filter. The antibiotics depend on the selection of the plasmid-encode genes utilized for the protein expression. 3. Streak a single colony of the bacteria transporting the plasmid in a LB?+ antibiotic plate and incubate for 18?h at 37C. Key resources table We suggest using molecular biology grade reagents and to autoclave and filter all the following buffers. Rich medium, glucose and ampicillin 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 usually a base and caustic material. It is usually highly recommended to wear protecting gloves, clothing, vision, and face protection. Prepare immediately Sulfacarbamide before use, total, EDTA-free protease inhibitor cocktail dilute to 1 1 and 1?M Maltose. Column buffer Sterilize with a 0.22?m filter. Elution buffer Prepare immediately before use. NuPAGE? MES SDS running buffer Prepare immediately before use. Transfer buffer BIO-RAD Prepare immediately before use. CRITICAL: Methanol is Sulfacarbamide usually a wood alcohol highly flammable and harmful. Avoid contact with skin and eyes. Avoid inhalation of vapor and keep away from sources of ignition. For the affinity purification of antibodies, prepare PBS – Phosphate-buffered saline 1?+ 0.1% TWEEN? 20 and a 0.2?M of glycine answer adjusted to pH 2.6 with HCl and sterilize with a 0.22?m filter. The 5% bovine serum albumin answer should be freshly made and keeping at 4C until use. CRITICAL: Glycine is usually a proteinogenic amino acid that may cause irritation. Avoid contact with eyes, skin, and clothing. CRITICAL: Hydrochloric acid also known as muriatic acid, is usually a corrosive material and harmful if inhaled. Handle the reagent while under a fume hood, wear a chemical-resistant apron, gloves, and goggles. Washing buffer Dynabeads Sterilize with a 0.22?m filter. Extraction buffer (Option A) For this protocol we used the pDESTH1 vector (Ampicillin resistant). 1. Add 5?mL LB medium, 0.2% glucose, and 10?L Ampicillin (stock 50?mg/ mL) to a culture tube. 2. Inoculate with a single bacterial colony from your agar plate (from day 0). 3. Culture at 37C and shake for 16 h. The LB medium can be stored at 20CC25C for 1?month. 50% glucose can be stored at 4C for 6?months and Ampicillin should be store at -20 for up to 6?months. Protein induction Timing: 5C6 h; day 2 4. In an Erlenmeyer flask, inoculate 500?mL of rich medium glucose (0.2%) and Ampicillin (100?g/ mL) with 5?mL of a culture of cells grown for 16?h.