Criterion blotter wet transfer

Pure samples of apoE2, apoE3, apoE4 (2 µg each), and Aβ42 peptide (100 ng) were resolved on 16.5% Tris-Tricine SDS gels (Bio-Rad 4563063) using 1x Tris-Tricine running buffer (Bio-Rad 1610744). The proteins were then transferred onto 0.2-µm nitrocellulose membranes using a Bio-Rad Criterion blotter (Wet transfer) for 20 min at 100 V. The membrane was then incubated with 2% formaldehyde in PBS for 30 min to fix Aβ42 peptide to the membrane. Next, the membranes were blocked with 5% milk in 1xTBST for 1 hr at room temperature and incubated at 4 °C overnight with 6E10 antibody (BioLegend, Cat. No. 80300) and anti-apoE primary antibody (Abcam, Cat. No. ab183597) at 1:1000 and 1:2000 dilution, respectively. Secondary antibodies, Alexa Fluor® 680 Anti-Rabbit IgG (Jackson ImmunoResearch Laboratories, Cat. No. Inc 711-625-152), and Alexa Fluor® 790 Anti-Mouse IgG (Jackson ImmunoResearch Laboratories, Cat. No. 715-655-150) respectively were used at 1:50,000 dilution in 1xTBS-T (0.1% tween-20 in tris-buffered saline) for 1 hr at room temperature to stain the membranes. The membranes were washed with TBS-T for 10 min three times after primary and secondary antibodies incubation. Finally, proteins were visualized with an Odyssey XF Imager (LI-COR).

Adipose tissues were homogenized in Pierce IP Lysis Buffer (87787, ThermoFisher Scientific) containing complete EDTA-free Protease Inhibitor Cocktail (04693132001; Roche, Basel, Switzerland) and PhosSTOP (04906845001; Roche), and then centrifuged at 4 °C, 16000 ×g for 20 min. Using Pierce BCA Protein Assay Kit (23225; Thermo Fisher Scientific, Waltham, MA, USA), the samples were diluted and mixed with 4X Laemmli Sample Buffer (1610747; Bio-Rad, Hercules, CA, USA) and 10 mM dithiothreitol (DTT) to give a final concentration of 2 µg/µL. As for the cell experiment, 3T3-L1 cells were rinsed once with PBS and collected directly in 1X Laemmli Sample Buffer plus 10 mM DTT after treatment. All harvested samples were immediately boiled at 98 °C for 10 min and then stored at −20 °C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to separate proteins with different molecular weights, depending on the target protein’s size. Precision Plus Protein Dual Color Standards (1610374; Bio-Rad) and protein samples were loaded into gel wells. Electrophoresis was performed using running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS), and the voltages were set at 95 V and 110 V according to the sample during the stacking and resolving stages.
After electrophoresis, the separated proteins were transferred onto methanol-pre-wetted and transfer buffer-pre-equilibrated PVDF membrane (1620177; Bio-Rad) via the transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). The protein size was determined by either Trans-Blot Turbo Semi-dry Transfer System (170-4155; Bio-Rad) or Criterion Wet Transfer Blotter (170-4070; Bio-Rad). The proteins were transferred at 25 V constant voltage with 0.4 A for 35 min for semi-dry transfer. For wet transfer, gels were equilibrated in transfer buffer for 15 min to remove excessive SDS, and then the proteins were transferred at 70 V with 250 mA for 1 h.
Polyvinylidene fluoride (PVDF) membranes were washed with methanol and stained with Ponceau S solution to visualize the transferred proteins. The fragment membranes cut at the target position were blocked with 5% non-fat milk in tris buffered saline with tween (TBST) and incubated with target protein-specific primary antibodies (Table 1), which were diluted with 1% BSA in TBST overnight at 4 °C. Membranes were washed with TBST three times for 15, 10, and 10 min each and then incubated with the species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2500 to 1:5000 diluted with 5% non-fat milk in TBST) for 1 h at room temperature. Membranes were washed with TBST three times for 10, 5, and 5 min each, and then the blotting images were visualized with Bio-Rad (1705061) and GE (RPN2235) enhanced chemiluminescence (ECL) substrates reagents using a Bio-Rad ChemiDoc Touch Imaging System. Blotting quantification was performed using Bio-Rad Image Lab software. Table 1 presents details about the antibodies.