WT splenic CD4 T cells, Jurkat cells, and HEK293T cells stimulated with LPS ± DAPT or transfected with the specified plasmids encoding V5-tagged NICD, HA-tagged STING, STING-β, STING-MRP, STING-Δ342, STING-Δ354, or STING-Δ368 were lysed in Pierce IP Lysis/Wash Buffer (Thermo Fisher Scientific) complemented with 1× Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). The cell lysates were then subjected to immunoprecipitation using antibodies against V5 (Cell Signaling Technology) for transfected proteins or using anti-STING and anti-NICD (Cell Signaling Technology) antibody for endogenous STING or NICD proteins. The antibody-antigen complex was precipitated using Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific). Immunoprecipitated complexes were eluted from the magnetic beads with an elution buffer and a neutralization buffer (Thermo Fisher Scientific). The sample was resolved on a 4 to 20% SurePAGE bis-tris precast gradient gel and blotted for the respective proteins.
Pierce ip lysis wash buffer
Manufactured by Thermo Fisher Scientific
Pierce IP Lysis/Wash Buffer is a solution designed for use in immunoprecipitation (IP) experiments. It is a lysis and washing buffer that helps to extract and purify target proteins from cell or tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Elution buffer, by Thermo Fisher Scientific (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Pierce protein a g magnetic beads, by Thermo Fisher Scientific (1 mentions)
Surebeads protein a magnetic beads, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Pierce crosslink magnetic ip co ip kit, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Cell Signaling Technology (1 mentions)
Rabbit igg isotype control, by Cell Signaling Technology (1 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Westsave up, by AbFrontier (1 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
Protease inhibitor cocktail 1, by Merck Group (1 mentions)
Gapdh, by AbFrontier (1 mentions)
4 protocols using pierce ip lysis wash buffer
1
Profiling STING and NICD interactions
2
Protein Extraction and Immunoprecipitation
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Cells were lysed in 500 μl Pierce™ IP lysis/wash buffer (ThermoFisher) with protease inhibitor cocktail (Sigma) on ice for 15 min. 350 μg of protein was used with SureBeads™ Protein A Magnetic Beads (Bio‐Rad) per manufacturer's instructions, using 10 μg antibody or corresponding normal IgG control (SCBT).
3
Co-Immunoprecipitation of PAK1 Protein Complexes
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Co-IP was carried out using the Pierce Crosslink Magnetic IP/Co-IP Kit (Thermo Fisher), adhering to the provided instructions. PAK1 primary antibody (#2602, Cell Signaling Technology) or Rabbit IgG isotype control (#2729, Cell Signaling Technology) (10 μg) were covalently cross-linked to 25 μL of Protein A/G Magnetic Beads (Thermo Fisher). Protein extraction was performed using Pierce IP Lysis/Wash Buffer (Thermo Fisher), supplemented with PMSF (Cell Signaling Technology) and a protease and phosphatase inhibitor cocktail (Thermo Fisher). A fraction of each sample was reserved as input. Equal amounts of protein extracts (1 mg) were incubated with the antibody- or IgG-cross-linked Protein A/G Magnetic Beads overnight at 4°C. Post-incubation, the beads were washed to eliminate unbound material, and proteins were eluted using a low-pH elution buffer, which facilitates the dissociation of the antigen from the antibody-bead complex. The eluates were neutralized with Neutralization Buffer and prepared for SDS-PAGE and Western blotting by adding Lane Marker Sample Buffer containing β-mercaptoethanol.
4
Western Blot: Comprehensive Protein Detection
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Western blot was performed as follows. Briefly, whole cell lysates prepared in ice-cold Pierce IP Lysis/Wash Buffer (Thermo Fisher Scientific, 87787) containing protease inhibitor cocktail I (Sigma-Aldrich, P8340), and phosphatase inhibitor cocktail III (Sigma-Aldrich, P0044) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membranes (Immobilon-P; Millipore IPVH 08130), blocked with 5% skim milk (BD Biosciences, 232100) in 0.01 M TBS (pH 7.5; Welgene, ML023-03) containing 0.5% Tween 20 (TBST; Biopure, 56-40-6) and blotted with the indicated antibodies. Then, the membranes were reacted with horseradish peroxidase-conjugated goat anti-rabbit-IgG (1:5000; AbFrontier, LF-SA5002a) for 2 hours at room temperature and visualized by WESTSAVE up (AbFrontier, LF-QC0101).
Primary antibodies used for Western blotting were as follows: TIPRL (1:5000, Bethyl, A300-663A), CD133 (1:1000; CST, 5860), LC3B (1:1000; Sigma-Aldrich, L7542), HA-Tag (1:1000, CST, 3724 s) and GAPDH (1:10,000; AbFrontier, LF-PA0212).
Primary antibodies used for Western blotting were as follows: TIPRL (1:5000, Bethyl, A300-663A), CD133 (1:1000; CST, 5860), LC3B (1:1000; Sigma-Aldrich, L7542), HA-Tag (1:1000, CST, 3724 s) and GAPDH (1:10,000; AbFrontier, LF-PA0212).
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