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6 protocols using revert total protein stain solution

1

Protein Extraction and Western Blot

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Cells and brain tissue were lysed in ice-cold RIPA 1× (cat no 20-188, Merck), supplemented with protease inhibitors. Samples were spin at 12,000 × g for 15 min at 4 °C. The supernatant was collected, and protein concentration was determined via BCA Protein Assay (cat no 23227, ThermoFisher Scientific) following manufacturer’s instructions. For Western blot, 5–30 μg protein were separated on pre-casted 4-15% Mini-PROTEAN TGX Gels (cat no 4561086 and 4561084, Bio-Rad) and transferred onto nitrocellulose membranes (Trans-Blot Turbo Midi NC Transfer, cat no 170-4159, BioRad) using a semi-dry transfer protocol. Before blocking, the total amount of protein per lane was determined using REVERT Total Protein Stain Solution (cat no 926-11011, Li-Cor) and used for normalization. Blocking was performed in 3% BSA in TBST (0.02 M Tris base, 0.15 M NaCl, 0.05% Tween-20 in milliQ water, pH 7.2–7.4) for 1 h at RT and incubated overnight at 4 °C with the primary antibodies diluted in blocking buffer. Membranes were then washed 3× with TBST for 5 min at RT and incubated with secondary antibodies (IRDye, 1:15000 in blocking buffer, Li-Cor). After 3 washes with TBST, membranes were imaged with the Li-Cor Odyssey system. Uncropped blots are provided with this paper as Source Data.
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2

Western Blot Analysis of Protein Extracts

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Protein extracts were performed by lysing cells in NP-40 lysis buffer (1% NP-40, 20 mM Tris pH 8, 137 mM NaCl, 10% glycerol, 2 mM EDTA), containing protease and phosphatase inhibitor (Thermo Scientific). Protein extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes (BIO-RAD). In some experiments, equal protein loading was controlled by staining the membrane after transfer using REVERT total protein stain solution (LI-COR). Membranes were blocked in TBS 5% milk and probed with primary antibodies overnight, washed 4 × 5 min in TBS + 0.1%Tween before incubating with secondary antibodies conjugated to AlexaFluor680 or IRDye800 for 1 h, washed and read on Odyssey CLx imaging system (LI-COR). Proteins were quantified using ImageStudiolite software. When Revert staining (LI-COR) was used, Revert signal from a whole protein lane was considered for normalization. The antibodies are listed in Supplementary Data 5. The original uncropped images of all western blots are shown in Supplementary Fig. 7.
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3

Western Blot with Odyssey Imaging

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Proteins were purified, separated and blotted as described with modifications (Frenk et al., 2014 (link)): aged MEP cells were re-suspended in Laemmli buffer containing 100 mM DTT and separated on 15% acrylamide gels, and Odyssey blocking buffer PBS (LI-COR 927 – 40000) was used for antibody hybridisations. Membranes were probed with antibodies listed in Table S7 at given dilutions before imaging on a LI-COR Odyssey 9120 imager. Total protein detection was performed by staining membranes with REVERT Total Protein Stain solution (LiCor 926 – 11010) after antibody detection.
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4

Western Blot Protein Analysis Protocol

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Cells were extracted in NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH 8, 137 mM NaCl, 10% glycerol, 2 mM EDTA), containing protease and phosphatase inhibitor (Thermo Fisher Scientific). Extracts separated by SDS-PAGE were transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked in TBS 5% milk and probed with primary antibodies (1 h to overnight), washed 4 × 5 min in TBS 0.1% Tween-20 before incubating with secondary antibodies conjugated to AlexaFluor680 or IRDye800 for 1 h, washed and read on Odyssey CLx imaging system (LI-COR). Proteins were quantified using ImageStudiolite software. In some experiments, equal protein loading was controlled for by staining the membrane after transfer using either Ponceau-S or Revert total protein stain solution (LI-COR). The quantified Revert signal from a whole lane was used for normalization. Antibodies used for Western blotting are listed in Table S2.
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5

Immunoblot and Immunofluorescence Antibody Protocols

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All C9ORF72 antibodies are listed in Supplementary file 1. SMCR8 antibody is from Abcam (ab202283). WDR41 antibody is from Abgent (AP10866B-EV). TOM20 antibody is from Santa Cruz Biotechnology, Inc (sc-11414). PDI antibody is from Abcam (ab2792). GAPDH antibody is from OriGene (TA802519). LAMP1 antibody used for immunoblot is from Cell Signaling Technology (#9091). LAMP1 antibody used for IF in RAW264.7 cells is from Thermo (PA1-654A). Peroxidase-conjugated goat anti-mouse and anti-rabbit are from Jackson ImmunoResearch Laboratories. Odyssey IRDye 800CW goat anti-mouse-HRP, the REVERT total protein stain solution and the Odyssey Blocking buffer (TBS) are from LI-COR Biosciences. Alexa Fluor 647-conjugated mouse and rabbit secondary antibodies are from Invitrogen.
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6

Western Blot Analysis of Cellular Proteins

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Total protein was isolated from cells in RIPA lysis buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 1% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS) and quantitated using BCA protein assay reagents (Thermo Fisher Scientific), then separated on 4–12% NuPAGE Bis‐Tris gels (Life Technologies) and transferred to Immobilon‐FL membrane (MilliporeSigma, Burlington, MA, USA). Blots were stained using REVERT Total Protein Stain solution (LI‐COR, Lincoln, NE, USA) and probed with primary antibodies at 4°C overnight. Antibodies are detailed in Supplemental Table S2. Bound primary antibodies were detected using DyLight 680 and 800 labeled secondary antibodies (Thermo Fisher Scientific, 1:15,000) and scanned and quantified using the Odyssey (LI‐COR). Where possible, phospho‐ and total protein antibodies from different host species were used at the same time to image dual fluorescent signals. When not possible, or to examine other signaling proteins, blots were stripped with Restore Fluorescent Western Blot Stripping Buffer (Thermo Fisher Scientific) and then reprobed. Quantified values for each signaling molecule on individual membranes were normalized to the associated no‐flow, no‐ISO value at the earliest time point on that membrane.
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