Oxyblot kit

Renal tissues were homogenized in 10 volumes of homogenization buffer [50 mmol/l Tris/HCl (pH 7.4), 1 mmol/l EDTA, 1%(v/v) Nonidet P40, 150 mmol/l NaCl, 0.25% sodium deoxycholate and 1∶100 dilution of protease inhibitor cocktail (Sigma-Aldrich)]. The protein concentration in the samples was determined by the Bradford assay with BSA as a standard. For detection of protein oxidation, an Oxyblot kit (Millipore) was used. The carbonyl groups in proteins were first derivatized with DNPH (2, 4-dinitrophenylhydrazine) in the presence of 6% (w/v) SDS. The kit used is sensitive to detect as little as 10 fmols of dinitrophenyl residues. To determine the specificity, the oxidized proteins provided by the kit were included as a positive control. Treatment of samples with a control solution served as a negative control for the DNPH treatment. The reaction was stopped after incubation for 15 min at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Millipore, US).

Quantification of protein carbonyl levels was performed using an OxyBlot^® kit according to the manufacturer's protocol (S7150, Millipore, Île‐de‐France, France). Cytosolic protein extracts from soleus and gastrocnemius (15 μg) were derivatized with 1% DNPH for 15 min at RT and then separated on 10% SDS‐PAGE. After transfer, membranes were blocked successively incubated in 1% BSA in PBS/0.05% Tween 20 (PBST) during 1 h and then overnight at 4°C with anti‐DNP primary antibody (1:200) supplied in the OxyBlot kit. Thereafter, membranes were washed with PBST and incubated for 1 h at room temperature with infrared dye‐conjugated secondary antibodies (LI‐COR Biosciences). After being washed, blots were captured using the Odyssey Imaging System (LI‐COR Biosciences).

Carbonylated proteins were detected by the OxyBlot Kit (Millipore, S7150) as previously described,^{24 (link)} by using 15 μg of total proteins that were resolved in 12% SDS-polyacrylamide gels.

Carbonylated proteins were isolated using derivatization of protein carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) in the OxyBlot kit (Millipore) and detected using immunodetection. This was performed with a primary antibody directed against dinitrophenylhydrazone, using 25 μg per lane of total protein extract from ftsh4-1 and WT plants. The carbonylated proteins were visualised with the WesternBright™ Quantum Western Blotting Detection Kit (Advansta) and densitometry analyses performed using IMAGEQUANT software (Molecular Dynamics).

The presence of carbonyl groups in the protein side chains of crude protein extracts was determined by gel electrophoresis and Western blotting using Millipore’s OxyBlot kit (Catalog No. S7150) [23 (link)]. Cell pellets from fresh fermentation samples of pAB828 cultures controlled at 30% or 300% dO₂ were washed and disrupted by sonication in the presence of 50 mM of DL-Dithiothreitol as described above. After determining protein concentrations, 8 μg of total protein were used for derivatization with 2,4-dinitrophenyl hydrazine (DNPH) following manufacturer’s instruction, and a parallel sample was used as negative control by substituting the derivatization-control solution for the DNPH Solution. Derivatized samples and negative controls were separated by SDS-PAGE and transferred onto nitrocellulose membranes as previously described above. Membranes were incubated for 1 h in blocking/dilution buffer (1% BSA/PBS-T) on a rocker at room temperature, followed by 1 h in the primary antibody solution (Rabbit Anti-DNP diluted 1:150). After two rinses with 1X PBS-T, the membrane was incubated for 1 h in the secondary antibody solution (Goat Anti-Rabbit IgG HRP-conjugated diluted 1:300). The SuperSignal West Pico Chemiluminescent Substrate detection kit (Thermo Scientific, Rockford, IL) was used for signal development.

Carbonylated proteins were detected using the OxyBlot Kit (Millipore, S7150) as previously described.^{50 (link)} Briefly, 10 μg of total proteins were reacted with 2,4 dinitrophenylhydrazine (DNP) for 15 min at 25 °C. Samples were resolved on 10% SDS-polyacrylamide gels and DNP-derivatized proteins were identified by western blot analysis using an anti-DNP antibody and an appropriate horseradish peroxidase-conjugated secondary antibody.