CD8+ T cells from healthy donors were pelleted via centrifugation at 3,000 rpm for 15 minutes and then lysed in NP-40 buffer (20 mmol/L Tris-CL, pH 8.0, 137 mmol/L NaCl, 10% glycerol, 1% NP-40, 2 mmol/L EDTA) and concentrations measured via protein assay using Bio-Rad reagent (catalog no. 500-0006). Once diluted to equal concentrations, samples were combined with Laemmli sample buffer (Bio-Rad, catalog no. 1610747) and run using a Mini-PROTEAN Electrophoresis and Transfer system (Bio-Rad, catalog no. 1658004). Membranes were incubated with primary antibody overnight at 4°C (antibodies used were specific for NKG7, β-actin, ETS-1, or GAPDH; details listed in Supplementary Table S3). Secondary antibodies were added the next morning for 1 hour [horseradish peroxidase (HRP)–conjugated anti-rabbit and anti-mouse; details in Supplementary Table S3]. Signal was visualized using SuperSignal West Pico PLUS Chemiluminescence Substrate Kit (catalog no. 34577, Thermo Fisher Scientific) on a Syngene G:Box Chemi XX6 system running GeneSys software (V1.6.1.0; RRID:SCR_015770). Images were formatted for figures using Microsoft PowerPoint (Microsoft Office 2016). Full images are included in the Supplementary Data File S1.
Supersignal west pico plus chemiluminescence substrate kit
Manufactured by Thermo Fisher Scientific
The SuperSignal West Pico PLUS Chemiluminescence Substrate Kit is a laboratory reagent designed to enable chemiluminescence detection of proteins in Western blot applications. The kit provides a chemiluminescent substrate solution that can be used with horseradish peroxidase-conjugated detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Specific primary anti gfp polyclonal antibodies, by Merck Group (2 mentions)
Goat anti mouse antibodies coupled to horseradish peroxidase, by Vector Laboratories (2 mentions)
Mini protean electrophoresis and transfer system, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Milchpulver, by Carl Roth (1 mentions)
4 protocols using supersignal west pico plus chemiluminescence substrate kit
1
Western Blot Profiling of CD8+ T Cells
2
EV Protein Immunoblotting Procedure
3
Western Blot Analysis of Fluorescent Proteins
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All the samples obtained during the FLAG Pull-down assay were separated by SDS-PAGE using 12% gels and transferred to the Whatman Protran nitrocellulose transfer membrane (Protran BA85, Schleicher & Schuell Pure, Sigma-Aldrich) in the semi-dry system at 10 V for 40 min in Towbin buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3). The membranes were blocked at room temperature with 2% milk powder (Milchpulver, blotting grade, Roth) in the PBS buffer and incubated for 1 h at room temperature. Next, the membrane was incubated overnight at 4 °C with the specific primary anti-GFP polyclonal antibodies (Sigma-Aldrich) (diluted 1:300 with milk buffer), which cross-react with CFP and YFP. After washing (PBS supplemented with 0.02% Tween, 3 × 10 min), the membrane was incubated for 2 h with secondary goat anti-mouse antibodies coupled to horseradish peroxidase (Vector Laboratories, dilution 1:10000 with milk buffer). Specific signals were detected using the SuperSignal™ West Pico PLUS Substrate Chemiluminescence kit (Thermo Scientific™) according to the manufacturer’s manual. Finally, the membranes were exposed to Kodak BioLight film.
4
Western Blotting of Fluorescent Fusion Proteins
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All the samples obtained during the FLAG Pull-down assay were separated by SDS-PAGE using 12% gels and transferred to the Whatman Protran nitrocellulose transfer membrane (Protran BA85, Schleicher & Schuell Pure, Sigma-Aldrich) in the semi-dry system at 10 V for 40 min in Towbin buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3). The membranes were blocked at room temperature with 2% milk powder (Milchpulver, blotting grade, Roth) in the PBS buffer and incubated for 1 h at room temperature. Next, the membrane was incubated overnight at 4 °C with the specific primary anti-GFP polyclonal antibodies (Sigma-Aldrich) (diluted 1:300 with milk buffer), which cross-react with CFP and YFP. After washing (PBS supplemented with 0.02% Tween, 3x10 min), the membrane was incubated for 2 h with secondary goat anti-mouse antibodies coupled to horseradish peroxidase (Vector Laboratories, dilution 1:10 000 with milk buffer). Specific signals were detected using the SuperSignal™ West Pico PLUS Substrate Chemiluminescence kit (Thermo Scientific™) according to the manufacturer's manual. Finally, the membranes were exposed to Kodak BioLight film.
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