Anti mouse igg antibody

Cells were grown until half of the glucose was consumed and lysed in lysis buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM MgCl₂, 0.1% NP40] supplemented with 0.1% protease inhibitor cocktail (Calbiochem, USA) and 1 mM PMSF using acid-washed glass bead. After repeating 30 s on/90 s off cycle for 10 times, cell debris were centrifuged down for 20 min and total protein concentration of the supernatant was analyzed by Bradford assay. 800 μg of proteins were used for TAP pull down with 20 μL of IgG Sepharose 6 Fast Flow resin (GE healthcare, USA) for 1 h, washed three times with lysis buffer, and eluted by boiling with 5× sample buffer. Samples were resolved by size on 6% SDS-PAGE gel and analyzed by western blotting with anti-DDDDK antibody (MBL life science, USA) for flag tag and anti-mouse IgG antibody (Sigma-Aldrich, USA) for TAP tag. Blotted membrane was treated with proper HRP-conjugated secondary antibody and visualized by G::box Chemi-XL (Syngene, USA).

Cells were fixed with 4% paraformaldehyde (PFA) dissolved in deionized water for 15 min and permeabilized in PBS containing 0.1% Triton X-100. Following rinsing twice with phosphate buffer saline (PBS) containing 0.5% Tween 20 (PBST), cells were blocked in PBS containing 5% goat serum for 30 min. Cells were incubated with the primary rabbit anti-TRPM2 antibody (Bethyl) at a dilution of 1:1500 or mouse anti-PAR antibody (Enzo;1: 500) overnight at room temperature and, after extensive washing in PSBT, incubated with the secondary FITC-conjugated goat anti-rabbit IgG antibody (Sigma; 1:1000) or anti-mouse IgG antibody (Sigma; 1: 1000) for 1 hr at room temperature. After washing with PBS and rinsing in water, coverslips were mounted using florescent mounting medium with 4′,6-diamidino-2-phenylindole (DAPI). All images were captures using an Olympus IX51 microscope, a digital camera and Cell^F software (Olympus). The intensity of the fluorescent was quantified using ImageJ and at least 75 cells were examined in each well.

The N. benthamiana leaf samples were harvested 48 h after infiltration. Total protein was extracted from powdered plant tissue using a phenol solution (0.5 M Tris pH 9.4, 50 mM EDTA, 0.7 M sucrose, 0.1 M KCl containing 2% ß-mercaptoethanol and complete protease inhibitors (Roche)). The protein concentration was measured using a Bradford assay following the manufacturer’s instructions (Bio-Rad). 0.1 mg protein of each sample was separated on Tris-Glycine SDS-PAGE gels and transferred to nitrocellulose membranes using an iBlot (semi-) dry blotting system from Life Technologies. The blotting membrane was blocked with 5% BSA (bovine serum albumin) in TBST solution (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. The membrane was incubated with 1:3000 diluted anti-GFP primary antibody (Sigma) for 3 h at room temperature. The membrane was washed with TBST solution and incubated with a 1:3000 dilution of anti-mouse IgG antibody (Sigma). The signal was detected using an AP conjugate substrate kit (Bio-Rad).

Protein lysates (30 μg) were separated in 12% SDS-polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBS-Tween buffer for 1 h and incubated with primary anti-LC3B (1:2000 Novus, NB100-2220), anti-SQSTM1/p62 (1:1000 Cell signaling, 88588), and anti-β-actin (1: 5000 Sigma-Aldrich, A5441) antibodies at 4°C overnight. Subsequently, three washes were performed with 0.05% TBS-Tween for 10 min and incubated in secondary anti-Mouse IgG antibody (1:20,000 Sigma, A2304) or anti-Rabbit IgG (1:20,000 Sigma, A0545) for 1 h at room temperature. Finally, immunodetection was performed with HRP-Western chemiluminescent Immobilon Substrate (Millipore, WBKLS0500) on C-DiGit transfer scanner (LI-COR). Relative intensity of bands was obtained by densitometric analysis of four independent experiments using Image J software.

Two fractions of thylakoid membrane and stroma of chloroplasts were separated by differential centrifugation following an osmotic shock to intact chloroplasts. SDS-PAGE with 12% (w/v) acrylamide gels was used to separate the proteins in the fractions and the samples were loaded on the total protein basis (5 µg protein). Antibodies that were raised against RCA were used to detect the RCA proteins in western blotting after the polypeptides had been electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, USA). RCA antibodies were produced while using prokaryotically expressed products as antigens of RCA genes that were derived from O. meyeriana and the cultivar rice, respectively. After the proteins were transferred to the PVDF membrane, the secondary anti-mouse IgG antibody conjugated with alkaline phosphatase (Sigma) and the nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) system were used to detect and visualize the immunoblotting signals.