Odyssey blocking buffer obb

To detect the presence of antibody-cytokine chimera in transfection supernatant, approximately 10µL of sample was run on NuPAGE™ 4-12% Bis-Tris gels (Thermo Fisher Scientific) in MOPS buffer. Briefly, all samples were reduced by heating samples in the presence of sample LDS buffer and sample reducing agent (Thermo Fisher Scientific) for 10 minutes at 70°C before loading onto the gel. Subsequent to electrophoresis, samples were transferred to a PVDF membrane and blocked for 1 hour at room temperature (RT) with Odyssey Blocking Buffer (OBB; LI-COR). Membranes were stained using 1:1000 IRDye 800CY goat anti-mouse IgG (LI-COR) formulated in OBB with 0.1% Tween-20 and 0.1% SDS at RT for 1 hour. Subsequently, membranes were washed with 0.05% Tween-20 in PBS before a PBS rise. Membranes were scanned using the LI-COR Odyssey CLx.

Recombinant COX-2 (Sino Biological Inc, Beijing, China, Cat. No. 12036-H08B) was mixed with Laemmli buffer (Bio-Rad Laboratories, Hercules, CA), PBS and DTT (Dithiothreitol, Merck, Cat. No. 646563) to a final concentration of 2.5 μg/ml. In total, 20 μl of this solution was applied on a 7.5% SDS-PAGE precast gel (Bio-Rad Laboratories, Cat. No. 4561025) together with WesternSure Pre-stained Chemiluminescent Protein Ladder (LI-COR Biosciences, Lincoln, NE, Cat. No. 926-98000) and the proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, Cat. No. 1704270). Odyssey blocking buffer (OBB, LI-COR Biosciences, Cat. No. 927-40000) mixed 1:1 with PBS was used as blocking solution before incubation with patient plasma diluted (1:2000) in 60% PBS, 40% OBB and 0.2% Tween 20. Mouse anti-human IgG, Fc Fragment Specific (HP6043) Peroxidase Conjugate (1:1000) (Merck Millipore, Burlington, MA, Cat. No. 411550), diluted in 60% PBS, 40% OBB and 0.2% Tween 20 was used to detect the autoantibodies using Clarity Western ECL Blotting Substrates (Bio-Rad Laboratories) and ChemiDoc MP Imaging System (Bio-Rad Laboratories).

Cells were lysed with SDS lysis buffer (1% SDS, 10 mM EDTA, 1x EDTA-free protease inhibitors) and protein concentration was determined by the bicinchoninic acid assay method (BCA, Pierce) according to the manufacturer protocol. Samples were boiled for 5 min after the addition of 6 ×x Laemmli buffer (9% SDS and 60% glycerol, 375 mM Tris-HCl pH 6.8, 0.015% Bromophenol blue, 12% β-mercaptoethanol). Proteins were separated for 2 hr on a 12% SDS-polyacrylamide gel using a Bio-Rad mini-PROTEAN electrophoresis apparatus, transferred to low-fluorescence PVDF membranes (Millipore) for 2 hr at 4°C at a constant 70 V in 25 mM Tris, 150 mM glycine, and 10% (vol/vol) methanol transfer buffer, blocked with Odyssey Blocking Buffer (OBB, LI-COR Biosciences) for 1 hr at room temperature, and probed with 1:1000 dilution of mouse monoclonal anti-SCD antibody (ab19862; Abcam) in OBB overnight at 4°C. After washing with PBS-T, membranes were incubated with fluorescent goat anti-mouse secondary antibodies (926-32210; LI-COR Biosciences) at 1:15000 in OBB for 1 hr at room temperature. Signal was detected using an Odyssey Infrared Imaging System (LI-COR Biosciences). To ensure antibody specificity, shRNA knockdown of SCD1 was performed with two independent shRNA constructs (V3LHS_305870 and V3LHS_305872; Open Biosystems) and compared with non-silencing shRNA construct (RHS4346; Open Biosystems).