5 bromo 4 chloro 3 indolyphosphate

5-bromo-4-chloro-3`-indolyphosphate (BCIP), alkaline phosphatase-labelled anti-mouse secondary antibody, lysozyme, nitro-blue-tetrazolium (NBT) reagent, paraoxon-ethyl, protease inhibitor cocktail, urea, m-cresol purple, δ-valerolactone and tergitol NP-10 were purchased from Sigma–Aldrich, Bangalore, India. Mouse anti-PON1 primary antibody and 5-thiobutyl butyrolactone (TBBL) was a kind gift from Dr. Richard W James, University of Geneva, Switzerland and Dr. Tawfik D.S, Department of Biological Chemistry, Weizmann Institute of Science, respectively. Stratagene's Quick change site directed mutagenesis kit was purchased from Agilent Technologies, Gurgaon, India. Protein molecular weight markers and Bradford reagent were purchased from Bio-Rad, Gurgaon, India. DNA primers (oligonucleotides) used were synthesized and purchased from Sigma Aldrich, Bangalore, India. Chlorpyrifos oxon (CPO) and diisopropylfluorophosphate (DFP) were purchased from Chem. Service, Inc., West Chester, PA, USA. All other reagents used were of analytical grade. Buffers used were prepared in double distilled water.

Protein extraction and western blot analysis were performed as described previously [30 (link)]. Briefly, total proteins were extracted from 0.1-g leaf samples by grinding into fine powder in liquid nitrogen, followed by adding 300 µL of 2 × SDS buffer (100 mM Tris (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol, and 0.2% (w/v) bromophenol blue) with subsequent heating at 100 °C for 10 min. Proteins were separated in 12.5% SDS polyacrylamide gel by electrophoresis and transferred to nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) using a mini trans-blot electrophoretic transfer cell (Bio-Rad).
Western blot analysis was performed using the rabbit anti-flag antibody (Sigma-Aldrich, St Louis, MO, USA) diluted at 1:1000, or mouse anti-flag antibody (Sigma-Aldrich) diluted at 1:5000, or rabbit anti-GFP antibody (GenScript, Nanjing, China) diluted at 1:3000, or rabbit anti-PLRV movement protein (MP) antibody [45 (link)] diluted at 1:5000, as a primary antibody. Incubation was then performed with goat anti-rabbit IgG (Sigma-Aldrich) diluted at 1:3000, or goat anti-mouse HRP (Bio-Rad) diluted at 1:3000, as a secondary antibody. Finally, the membranes were detected using the nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyphosphate (Sigma-Aldrich), or with a superior chemiluminescence detection kit (GE Healthcare) according to the manufacturer’s instructions.