Advanblock chemi blocking solution

The molecular weight of purified PNPO samples analyzed by SDS‐PAGE was estimated by comparison with protein standards using the MS Image Capture‐A (Major Science, Taiwan) software. For the immunological detection of recombinant PNPO in cell lysates, aliquots of the bacterial cultures expressing either wild‐type or 1‐31del PNPO forms were withdrawn after 6 h from the addition of IPTG and centrifuged. Cell pellets were suspended in 250 μL of 20 mM potassium phosphate buffer, pH 7.6, and sonicated for 2 min with a 20‐s ON + 20‐s OFF cycle. SDS‐PAGE was performed on 20 μg protein samples, as estimated with a Bradford assay performed on lysates. Western blotting analyses were performed with a Bio‐Rad mini trans‐blot® system (BioRad, Hercules, CA, USA). The membrane was blocked for 1 h with the AdvanBlock™‐Chemi blocking solution (Advansta Inc., USA), incubated overnight at 4°C with an anti‐PNPO rabbit pAb (ABclonal, USA) diluted 1:1000 in TBST solution and then with a goat anti‐rabbit IgG (h/l chain) HRP‐conjugated secondary antibody (Bethyl Laboratories Inc., USA), at room temperature. Chemiluminescence was detected with a ChemiDoc™ Bio‐Rad Imaging system, after incubating the membrane with Pierce™ ECL (Thermo scientific Inc., USA).

Frozen kidney and liver tissues were crushed in liquid nitrogen using a Mixer Mill during 30 s at 30 Hz (MM400, Retsch technology), and 12 mg of tissue powder were lysed with 500 µL of RIPA solution (Ref#R0278, Sigma-Aldrich). The protein extracts were centrifuged for 20 min at 16,000 g and 4 °C, and protein concentration in the supernatant was determined using the BCA protein assay Kit (Ref#K812-1000, Biovision). Each protein sample (45 µg) was denatured and reduced in Laemmli buffer (62.5 mM TRIS pH 6.8, 2% SDS; 10% glycerol) containing 5% ß-mercaptoethanol before SDS-PAGE on a 4–15% polyacrylamide gel (Bio-Rad). Proteins were transferred onto a nitrocellulose membrane blocked with AdvanBlock-Chemi blocking solution (Advansta) during 1 h at room temperature. After washing in PBS-0.1% Tween 20, the membrane was incubated overnight at 4 °C with a rabbit polyclonal anti-MMUT primary antibody at 1/1000 in blocking solution (MUT Rabbit pAb, Ref#A3969, ABclonal). Revelation of the primary antibodies (after washing) was performed by incubation with goat anti-rabbit Horseradish peroxidase-conjugated secondary antibody (Ref#A0545, Sigma-Aldrick) at 1/10000 ratio in blocking solution for 1 h at room temperature, followed by enhanced chemiluminescence detection (WesternBright Quantum HRP substrate, Advansta) on a ChemiDoc Touch low-light camera (Bio-Rad) in automatic mode.