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Turboblot system

Manufactured by Bio-Rad
Sourced in United Kingdom

The TurboBlot system is a laboratory equipment designed for efficient and rapid protein transfer from a gel to a membrane during Western blotting analysis. It utilizes an innovative blotting technology to facilitate the transfer process.

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32 protocols using turboblot system

1

Cell signaling pathway analysis

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Cells were rinsed 3X with cold PBS and harvested in RIPA buffer containing protease (Complete Mini, Roche) and phosphates inhibitors (Thermo Scientific). Cells were incubated for 10 min (13) with ISO (50 nM), TLQP-21 (10 μM) or a combination of the two treatments. Lysates were then sonicated and centrifuged at 12,000 rpm for 10 min to remove nuclei. Protein was determined by BCA assay (Thermo Scientific) and an equivalent concentration of cell lysates were prepared in sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 min at 95°C. Proteins were resolved by a (4%–20%) SDS-polyacrylamide gel electrophoresis (Bio-Rad) and transferred to a PVDF membrane (Bio-Rad) by using a turbo blot system from Bio-Rad. Individual proteins were detected with the specific primary antibodies by overnight incubation at 4°C (tubulin #2146), pHSL (Ser660; #4126), pERK1/2 (Thr202/Tyr204, #9101) all from Cell Signaling at 1:1000 dilution. The HRP conjugated anti-rabbit antibody was used as a secondary antibody at 1:7000 dilutions and the blot was exposed to luminol enhancer solution by using ECL prime reagent (GE health care), further imaged using a chemidoc documentation system from Bio-Rad laboratories.
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2

Western Blot Protein Detection Protocol

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Cells were homogenized by scraping into 90 µl Urea/SDS buffer (10 mM Tris‐HCl pH 6.8, 6.7 M urea, 1% w/v SDS, 10% v/v glycerol and 7.4 µM bromophenol blue, containing 50 µM phenylmethysulfonyl fluoride and 50 µM N‐methylmaleimide, all Sigma Aldrich). Lysates were sonicated and 10% v/v β‐mercaptoethanol (Sigma Aldrich) added. Proteins were separated by SDS‐PAGE using 10% polyacrylamide gels; 1.5 M Tris, 0.4% w/v SDS, 10% acrylamide/bis‐acrylamide (all Sigma Aldrich), electrophoresis buffer; 25 mM tris‐HCl, 190 mM glycine, 0.1% w/v SDS, pH 8.5 (all Sigma Aldrich). Proteins were transferred to a nitrocellulose membrane using the TurboBlot system (BioRad) and blocked at room temperature in Odyssey® TBS‐Blocking Buffer (Li‐Cor BioSciences) for 1 h. The membranes were probed overnight at 4°C diluted in blocking buffer, washed 3 × 5 min in PBS with 0.1% Tween (both Sigma Aldrich) and probed for 1 h at room temperature in the dark with IRDye® conjugated secondary Abs against goat IgG (800 CW) and rabbit IgG (680 CW), raised in goat or donkey (LiCor BioSciences), diluted in Odyssey® TBS‐Blocking Buffer at 0.05 µg/ml. Membranes were then washed for 3 × 5 min in PBS with 0.1% Tween, rinsed with ddH2O and imaged using the Odyssey® CLX 9120 infrared imaging system (LiCor BioSciences). Image Studio Light v.5.2 was used to process scanned membranes.
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3

Western Blot Analysis of Eukaryotic Translation Factors

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Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing an inhibitor cocktail of phenylmethylsulfonyl fluoride (PMSF) (1:100) (American Bioanalytical), Protease Inhibitor Cocktail (Sigma-Aldrich) (1:100), and Phosphatase Inhibitor Cocktail I (Abcam) (1:100). Lysates were collected and clarified by centrifugation. The lysate was mixed with 4× Laemmli buffer, boiled, and then processed by SDS-PAGE. The samples were then transferred from the gel to a 20-μm nitrocellulose membrane (Bio-Rad) using the TurboBlot system (Bio-Rad). Membranes were blocked in 5% dry milk with 0.1% Tween-20 or Everyday Blocking Reagent (Bio-Rad) and probed with anti-eIF2A (1:1,000), anti-eIF2D (1:1,000), anti-p-eIF2α (1:1,000), anti-p-PKR (1:1,000), anti-eIF2α (1:2,500), or anti-PKR (1:2,500) antibodies overnight. Anti-rabbit/mouse immunoglobulin G antibody coupled to peroxidase (Amersham GE Healthcare) were used as secondary antibody at a 1:5,000 dilution. Protein bands were visualized with the ECL detection system (Amersham, GE Healthcare). Where stated, membranes were subjected to reprobing against anti-tubulin (1:5,000) or anti-GAPDH (1:5,000).
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4

Protein Extraction and Western Blot Analysis

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PECs were lysed in RIPA Buffer (20 mM HEPES, 100 mM NaCl, 5 mM EDTA, 1% Nonidet-P40+ Protease & Phosphatase Inhibitor Cocktail, Merck Life Science). Proteins were quantified by Bradford Protein Assay (Merck Life Science) and 30 μg of proteins were resolved on SDS-PAGE, transferred on nitrocellulose membrane by the TurboBlot system (BioRad, Hemel Hempstead, UK). Proteins were detected with primary antibodies and peroxidase-conjugated secondary antibodies (Bio-Rad) and resolved by chemiluminescence analysis using ECL (Thermo Fisher Scientific). Densitometry analysis was performed with the Image Lab program (Bio-Rad, Hemel Hempstead, UK). The list of primary antibodies used are listed under “Reagents.”
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5

Western Blotting of GFP Fusion Proteins

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Western blotting was performed using a standard trichloroacetic acid (TCA) protocol, exactly as described in (Chen et al., 2017 ), except using a mouse anti-GFP JL8 antibody (Clontech). In short, 5ml of meiotic cells in culture (or 2ml vegetative) were collected and incubated with 5% TCA for at least 10 minutes at 4°C. Cells were centrifuged for 2 min. at 20,000 rcf and supernatant was aspirated. Cells were washed with acetone and pellets dried for at least 2 hours. Cell extract was made by addition of TE, supplemented with 3 mM DTT and protease inhibitors (Roche), and 1 volume of acid-washed glass beads. Tubes were agitated for 5 minutes, after which 3X SDS sample buffer was added, samples were boiled for 5 minutes, centrifuged for 5 min at 20,000 rcf. 8 μl supernatant was loaded onto Bis-Tris acrylamide gels. Gels were transferred using a Turboblot system (BioRad). Primary anti-GFP antibody dilution was 1:2000, anti-hexokinase was 1:12,000, secondary (Li-Cor) was 1:20,000. Primary antibody incubation was overnight, secondary for 1–2 hours. Blots were visualized using a Li-Cor system.
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6

Western Blot Analysis of CEACAM and Opa

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All samples were boiled for 20 min and homogenized by passing through a needle (30 gauge) and syringe ten times. Twenty μL were loaded onto a pre-cast 4–20% acrylamide gel (Bio-Rad, Hercules, CA). After electrophoresis, gels were transferred to 0.2 μM nitrocellulose using the turbo blot system (Bio-Rad), and immunoblotted with the A0115 α-CEACAM antibody (Dako, Carpentaria, CA), or the 4B12 pan-Opa antibody (hybridoma generously provided by Christof Hauck, University of Konstanz, Germany, and antibody purified by the University of Virginia Hybridoma Core) followed by IRDye 800 CW conjugated goat anti-mouse or goat anti-rabbit IgG (LI-COR Biosciences, Lincoln, NE). Blots were visualized with an Odyssey imager (LI-COR Biosciences). NCCM pellet band intensities were measured using ImageJ51 analysis software, and plotted with Origin Pro 7.5. Quantification of NCCM relative to the total concentration of NCCM or Opa proteins is not feasible because of a lack of a loading control and antibody reactivity differences.
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7

Immunoblot Analysis of Rad53 Protein

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TCA-precipitated proteins were separated by SDS-PAGE using precast gels (Invitrogen), and blotted onto polyvinylidene difluoride membranes using a Bio-Rad Turbo blot system. Rad53 protein was detected using a custom-made mouse monoclonal antibody67 (link). Uncropped immunoblots are shown in Supplementary Fig. 9.
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8

Western Blot Protein Analysis Protocol

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Four microliter of the crude lysate or RNCs (ribosome pellet) (representing 0.4%) in 1 x NuPAGE loading dye (Invitrogen) and 10 µL of boiled beads (representing 4%) in 1 x NuPAGE loading dye were loaded onto NuPAGE Bis-Tris gels (MW < 100 kDa) (Invitrogen) or Tris-Glycine gels (Bio-Rad; MW > 100 kDa) and run at 160 V for 50 min. Protein was transferred onto 0.45 µm TransBlot Turbo nitrocellulose (Bio-Rad) using the High MW setting of the TurboBlot system (Bio-Rad) according to the manufacturer’s procedure. Membranes were blocked in 5% milk in TBS-T (0.02% Tween-20) for 1 hr at room temperature under gentle shaking. Primary antibody (1:5000) was added and incubated overnight at 4 °C while constant shaking. Next, membranes were washed in TBS-T and a secondary antibody (1:10,000) was applied. The membrane was stained for 1 h at room temperature. Before visualization by ECL developing solution (Bio-Rad), membranes were washed again. Membranes were imaged using the Chemidoc (Bio-Rad). Antibodies used in this study are listed in Supplementary Table 3.
Fas1- and Fas2-IPs were stained using Instant Blue (abcam) according to the manufacturer’s protocol. For Nsp1 pull-downs, NuPAGE Bis-Tris gels were stained using the SilverQuest Silver Staining Kit (Invitrogen).
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9

Western Blot of Neuronal Proteins

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For Western blot analysis Lcells, NCad-Venus Lcells and hippocampal neurons grown in 6 cm cell culture dishes were washed with PBS and lysed in 1.5 x LDS sample buffer (life technologies) supplemented with DTT (10 x reducing agent, life technologies) and protease inhibitors (Calbiochem). Lysate aliquots equivalent to 5 μg of total protein were loaded on 4 – 12 % BisTris NuPAGE gels (life technologies), separated by electrophoresis with MOPS running buffer (life technologies) and transferred to Immobilon-FL (Millipore) using the TurboBlot system (Biorad) at 25 V, 2.5 A for 7 min. Ponceau stain (Sigma) was used to validate homogeneous loading and transfer. Membranes were blocked for 1 h with Odyssey blocking buffer (Li-cor) and incubated overnight at 4 °C with primary antibodies in a 1 + 1 mixture of Odyssey blocking buffer and PBS containing 0.2 % Tween20. Membranes were rinsed several times with water followed by 2 × 10 min washes in PBS containing 0.2 % Tween20. Secondary antibodies coupled to IRdye680 and IRdye800 (Li-cor, 1:15.000) were applied for 1 h in a 1 + 1 mixture of Odyssey blocking buffer and PBS containing 0.2 % Tween20. Membranes were washed for 10 min in PBS containing 0.2 % Tween20 and 0.01 % SDS, 10 min in PBS containing 0.2 % Tween20 and rinsed several times in water before the signal was scanned using an Odyssey Infrared imager (Li-cor).
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10

Western Blot Analysis of Arg1 in Liver

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Liver tissues were homogenized in a solution containing T-PER solution and 1× HALT protease inhibitor cocktail (40 μl/mg tissue). Homogenates were further diluted in T-PER to a concentration of 1 mg/ml protein with 2x Laemmli buffer. Protein samples (20–30 μg) were subjected to Western Blot analysis. Thus, proteins separated by electrophoresis in 10% TGX FastCast acrylamide gels (Bio-Rad) were transferred to PVDF membrane (Bio-Rad, TurboBlot system) and probed with rabbit polyclonal anti-Arg1 (C-terminal peptide; 1:10,000 dilution; Abcam ab91279) and mouse polyclonal anti-β-actin (1:7500 dilution; Sigma) or anti-tubulin antibodies. Immunoreactive proteins were detected using HRP-conjugated goat anti-rabbit or anti-mouse secondary antibody (1:7500; Sigma) enhanced chemiluminescence signal. Digitized images were recorded with a FluorChem 8900 instrument (Alpha Innotech, San Leandro, CA). Some images underwent semi-quantitation with publicly available Image J software (Version 1.47, NIH).
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