Nupage novex bis tris mini gel

Western blots were conducted as described [8] (link). Pure protein and cell-free medium samples were incubated at 72°C for 10 minutes in 1x NuPage SDS loading buffer, then run on a 4%–12% NuPAGE Novex Bis-Tris mini gel (Invitrogen). The proteins were then transferred to a PVDF membrane (pore size, 0.45 µm; Amersham Hybond-P), blocked with 2% ECL Prime blocking agent (GE Healthcare) in PBST for 1 hour at RT, and washed three times with PBST (3 minutes each). Monoclonal antibodies were diluted to 1 µg/mL in blocking solution and incubated with the blots for 1 hour at RT, then the blots were washed thrice again in PBST. GAM-HRP antibody (Promega) at a 1/10,000 dilution was incubated on the blot for 1 hour at RT. The blots were washed four more times with PBST (5 minutes each), and developed using Lumigen TMA-6 (Lumigen) substrate. The blots were visualized with a 5 minute exposure using a FluorChem HD2 (Alpha Innotech). All westerns were conducted three times.

Western blots were conducted as previously described [16 (link)]. Pure toxin/toxoid were denatured at 72°C for 10 minutes in 1x NuPage LDS loading buffer, and then separated on a 4%–12% NuPAGE Novex Bis-Tris mini gel (Invitrogen). The proteins were transferred to a PVDF membrane (pore size, 0.45 μm), blocked with 2% ECL Prime blocking agent (GE Healthcare) in PBST, and washed with PBST (3x). Monoclonal or polyclonal antibodies were diluted to 1 μg/mL in blocking solution and incubated with the blots for 1 hour at room temperature. After washing (3x) in PBST, the blots were incubated with GAR-HRP or goat anti-mouse HRP (GAM-HRP) antibodies (Promega) at 5 ng/mL for 1 hour at RT. The blots were developed using Lumigen TMA-6 (Lumigen) substrate and visualized using a FluorChem HD2 (Alpha Innotech). All western blots were analyzed at least three times.

Cell lysates were prepared using a whole cell lysis buffer (20 mM Tris-Cl pH 7.6, 150 mM NaCl, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS). Nuclear and cytoplasmic fractionation was performed using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific), as per the manufacturers’ instructions. Protein samples were denatured at 90 °C for 10 min with 100 mM DTT in NuPAGE® LDS sample buffer. Samples were separated using a 4–12% NuPAGE® Novex® Bis-Tris mini gel (Invitrogen) and transferred onto PVDF membrane (Millipore) using Trans-Blot® SD Semi-Dry Transfer Cell (BioRad). Each blot was then probed with antibodies specific to the protein of interest (Additional file 2: Table S2).

A 1mm 10% NuPAGE^® Novex^® Bis-Tris Mini Gel was set up using the XCell SureLock Mini-Cell system (Invitrogen) as per manufacturer’s instructions for reduced samples. 50μg of mouse sperm from each of the caput, corpus, and cauda epididymis were loaded and the gel was run for 35 minutes at a 200V constant. Following electrophoresis, the gel was stained using SimplyBlue^™ SafeStain (Invitrogen) and destained as per manufacturer’s instructions. The gel was transferred to a gel slicer (built in house) and each of the 3 lanes was separated from the rest of the gel by cutting vertically. Each lane was then cut horizontally into 16 equal gel slices. Each gel slice was further cut into smaller (approximately 1x1x1mm) pieces which were transferred to a 0.6mL microcentrifuge tube (16 tubes per segment, for 48 total tubes) and were stored at -80°C until needed.

Western immunoblots were conducted as previously described [7 (link)]. Pure toxin/toxoid and cell-free medium samples were incubated at 72°C for 10 minutes in 1x NuPage SDS loading buffer, then run on a 4%–12% NuPAGE Novex Bis-Tris mini gel (Invitrogen). The proteins were then transferred to a PVDF membrane (pore size, 0.45 μm; Amersham Hybond-P), blocked with 2% ECL Prime blocking agent (GE Healthcare) in PBST, and washed with PBST (3x). Monoclonal antibodies were diluted to 1 μg/mL in blocking solution and incubated with the blots for 1 hour at RT, then the blots were washed again (3x) in PBST. GAM-HRP antibody (Promega) at a 1/20,000 dilution was incubated on the blot for 1 hour at RT. The blots were washed four more times with PBST (5 minutes each), and developed using Lumigen TMA-6 (Lumigen) substrate. The blots were visualized with a 5 minute exposure using a FluorChem HD2 (Alpha Innotech). All westerns were conducted three times.

Cortical neurons transduced with AAV.ULK1.DN and AAV.CTRL were lysed and the protein content of each sample was determined as described above. After precipitation with acetone, 50 µg of protein lysates per sample were separated on a 4–12% NuPAGE Novex Bis-Tris Minigel (Thermo Fisher Scientific). Following Coomassie staining, the protein areas were cut out, diced, and subjected to reduction with dithiothreitol, alkylation with iodoacetamide, and finally ON digestion with trypsin. Tryptic peptides were extracted from the gel, the solution dried in a Speedvac, and subjected to nanoLC-MS/MS as described previously [10 (link), 22 (link)]. Details of mass spectrometric analysis and data processing are provided in the SI.
Functional annotation of differentially regulated proteins to GO terms was performed in DAVID version 6.8 [21 (link)]. The functional annotation module was applied for GO biological process and cellular component terms using an EASE score of 0.1 and a minimum number of 2 counts. For analysis of protein–protein interaction networks, STRING version 10.5 [23 (link)] was used with a minimum required interaction score of 0.4. The STRING database’s k-Means clustering tool was employed to group proteins with roles in similar processes into four clusters.