CD8+ T cells from healthy donors were pelleted via centrifugation at 3000 rpm for 15 min and then lysed in NP-40 buffer (20 mM Tris-CL, pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP-40, 2mM EDTA) and concentrations measured via protein assay using BioRad reagent (cat. #500-0006). Once diluted to equal concentrations, samples were combined with Laemmli sample buffer (BioRad, cat. #1610747) and run using a Mini-PROTEAN Electrophoresis and Transfer system (BioRad, cat. #1658004). Membranes were incubated with primary antibody overnight at 4 °C (antibodies used were specific for NKG7, Beta-Actin, ETS-1, or GAPDH; details listed in Supplementary Table S3 ). Secondary antibodies were added the next morning for 1 hour (HRP-conjugated anti-rabbit and anti-mouse, details in Supplementary Table S3 ). Signal was visualized using SuperSignal West Pico PLUS Chemiluminescence Substrate Kit (cat. #34577, Thermo Fisher) 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 .
Mini protean electrophoresis and transfer system
Manufactured by Bio-Rad
The Mini-PROTEAN Electrophoresis and Transfer System is a laboratory equipment used for the separation and transfer of proteins or DNA/RNA molecules. It provides a platform for performing gel electrophoresis and Western blotting procedures. The system includes a casting stand, electrophoresis cell, and power supply to facilitate these common analytical techniques in a compact and efficient manner.
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2 protocols using mini protean electrophoresis and transfer system
1
Western Blot Analysis of Immune Cells
2
Western Blot Profiling of CD8+ T Cells
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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.
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