Mini protean tris glycine precast protein gels

Protein extracts were prepared by boiling cells in lysis buffer (125 mM Tris-HCl pH 6.8, 20% (w/v) glycerol, 4% SDS) for 10 minutes. Lysates were then mixed with Laemmli buffer and subjected to an additional boiling step. Approximately 15–20 μg of protein were loaded onto either 10% in-house acrylamide gels or 4–20% Mini-PROTEAN Tris-Glycine Precast Protein Gels (BioRad). Samples were then transferred to an Immobilon-FL Transfer PVDF Membrane (Millipore) for 2 hours at 4°C and 70V.
To block nonspecific binding, membranes were incubated in 5% BSA-TBS for 1 hour at room temperature. This was followed by an overnight incubation with primary antibodies at 4°C in 5% BSA-TBS with 0.1% Tween20. Afterwards, membranes were washed three times in TBS with 0.1% Tween20. And incubated with the corresponding IRDye-conjugated secondary antibody in 5% BSA-TBS with 0.1% Tween20. After three washes with buffer, membranes were dried and subjected to analysis using the Odyssey CLx system and ImageStudio Odyssey CLx software (LI-COR BIOSCIENCES, Lincoln, NE), following the manufacturer’s protocols.

For protein extractions, cell pellets were resuspended in RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40 y 1% sodium deoxycholate) supplemented with protease inhibitors and incubated on ice for 30 min with constant agitation. The lysate was then centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was sonicated using a Bioruptor (Diagenode, UCD-200) for 1 cycle of 3 minutes (high power, 30 s on, 30 s off). Protein concentration was determined by Bradford assay (Applied Biochem, A6932). 20 μg of protein was loaded into home-made 10% polyacrylamide gel with SDS or 4%–20% Mini-PROTEAN tris-Glycine Precast Protein Gels (Biorad, 4561096) and electroblotted onto Immobilon-FL Transfer Membranes (Millipore), after 5 minutes methanol activation. Membranes were then blocked in Odyssey Blocking Buffer (LI-COR Biosciences, 927-40000) for 1 hour and then probed with required primary antibodies for 2 hours. Membranes were washed with 0.1% Tween-20 - Odyssey Blocking Buffer and incubated with corresponding secondary antibodies (conc: 1:10.000) for 1 hour and finally, membranes were washed again with 0.1% Tween-20 - Odyssey Blocking Buffer. Membrane were analyzed using Odyssey CLx and ImageStudio Odyssey CLx Software (LI-COR BIOSCIENCES, Lincoln, NE) according to the manufacturer’s protocols.