A549 cells were cultured in 10 cm2 cell culture plates and treated with 1 mM diamide and 45 μM ALT in the presence or absence of 3 mM NAC for 4 h. Cells were collected and lysed in IP lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 0.1 mM PMSF, sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, and Leupeptin). 800 μg proteins from the clarified cell lysates were incubated overnight at 4 °C with rotation in the presence of STAT3 antibody (1:100 dilution). After incubation with sepharose A/G beads (Beyotime, Biotechnology) for another 1 h at 4 °C with rolling end-over-end, the immune complexes were collected, washed 3 times with cold lysis buffer and separated from the beads by boiling in LDS non-reducing sample buffer (ThermoFisher Scientific) for 5 min. The immunoprecipitated proteins were subjected to Western blotting for the detection of STAT3 and protein linked GSH (PSSG) using respective antibodies.
Lds non reducing sample buffer
Manufactured by Thermo Fisher Scientific
The LDS non-reducing sample buffer is a laboratory reagent used to prepare protein samples for analysis. It is designed to maintain the native structure of proteins without inducing disulfide bond reduction. The buffer contains lithium dodecyl sulfate (LDS) as the denaturing agent and is formulated to be non-reducing, allowing for the analysis of proteins under non-denaturing conditions.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using lds non reducing sample buffer
1
STAT3 Immunoprecipitation and Glutathionylation
2
Comprehensive Extracellular Vesicle Protein Analysis
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Purified sEV samples were incubated on ice in 10X RIPA buffer supplemented with protease inhibitor cocktail (Cell Signaling Technology, #9806) and proteosome inhibitor MG-132 (Sigma, #M7449). Either 4X reducing sample buffer or 4X LDS non-reducing sample buffer (Thermo Scientific, #84,788) was added to the sEVs before heating at 95°C for five minutes. sEV samples were electrophoresed on SDS-polyacrylamide gels and subsequently transferred to a methanol-activated Immobilon-P Transfer Membrane (#IPVH00010 Immobilon). After blocking at room temperature for one hour with blocking buffer (1X TBS pH 7.6 containing 0.05% Tween-20 and 5% fat-free dry milk), membranes were incubated at 4°C with rocking overnight using primary antibodies for the following proteins: ALIX (1/1,000, Proteintech #67,715-1-Ig), β-Actin (1/10,000, Santa Cruz Biotechnology, #sc-47778), CD-9 (1/1,000, Proteintech #20,597-1-AP), Flotillin 1 (1/1,000, Proteintech #15,571-1-AP), GAPDH (1/2,000, Cell Signaling Technology #5174), Hif-1α (1/1,000, Novus Biologicals #NB100-449), placental alkaline phosphatase (1/1,000, Boster Biological Technology #A01718), TSG101 (1/1,000, Proteintech #28,283-1-AP). Following incubation, membranes were subsequently washed and incubated with secondary antibody at 1/10,000 for 1 h at room temperature and visualized via West Pico SuperSignal chemiluminescence.
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