TMEM41B KO HAP1 cells reconstituted with full length TMEM41B fused to tagRFP (infected and uninfected) were collected and lysed in nonyl phenoxypolyethoxylethanol (NP-40) buffer (10 mM HEPES, pH 7.5, 150 mM KCl, 3 mM MgCl2, 0.5% NP-40) supplemented with cOmplete Mini EDTA-free protease inhibitor tablet (Millipore/Sigma: 11836170001) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) (Millipore/Sigma: 10837091001). Protein concentrations were determined by BCA assay as described above. Per sample, 50 μL (1.5 mg) Dynabeads Protein A for Immunoprecipitation (ThermoFisher Scientific: 10001D) were prepared and linked to 5 μg antibodies (rabbit anti-tagRFP and normal rabbit IgG control) according to the manufacture’s protocol. 100 μg of whole cell lysate (WCL) were incubated with beads-antibody complexes for 70 min at RT followed by wash steps according to the manufacture’s protocol. The resulting beads-antibody-antigen complexes were subsequently treated with elution buffer, mixed with NuPAGE® LDS Sample Buffer (ThermoFisher Scientific: NP0008) and NuPAGE® Sample Reducing Agent (ThermoFisher Scientific: NP0009) as per manufacturer’s instructions and denatured for 15 min at 70°C. The precipitated proteins within the eluted fractions were resolved by western blot as described above.
Dynabeads protein a for immunoprecipitation
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dynabeads Protein A for Immunoprecipitation are magnetic beads coated with Protein A, a bacterial protein that binds to the Fc region of immunoglobulins. These beads can be used to capture and isolate target proteins or protein complexes from biological samples for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Complete mini edta free protease inhibitor tablet, by Merck Group (1 mentions)
Page gel rapid preparation kit, by Ipsen (1 mentions)
Wj103, by Ipsen (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
Gel doc xr gel documentation system, by Bio-Rad (1 mentions)
Chemidoc xrst image system, by Bio-Rad (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
4 protocols using dynabeads protein a for immunoprecipitation
1
Immunoprecipitation of TMEM41B Protein
2
Fluorescent Antibody Staining and Protein Analysis
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Goat anti‐rabbit FITC fluorescent secondary antibody kit (BD5003, Bioworld, USA).
Goat anti‐rabbit TRITC fluorescent secondary antibody kit (BD5005, Bioworld, USA).
Goat anti‐mouse TRITC fluorescent secondary antibody kit (BD5004, Bioworld, USA).
DPP‐IV/CD26 (High‐purity dimer) protein (9168‐SE‐010, R&D systems, USA).
Lipofectamine™ 2000 transfection reagent (11668019, ThermoFisher, USA).
Dynabeads™ protein A for immunoprecipitation (10001D, ThermoFisher, USA) Fluorescent mounting tablets (including DAPI) (ZLI‐9557, Zhongshan Golden Bridge, China).
Western primary antibody diluent (ZS402‐1A, ZOMANBIO, China).
4% tissue cell fixative (P1110, Soleibao, China).
Foetal bovine serum (South America) (MP20002‐500 ml, Yuanye Biology, China).
RPMI 1640 medium (C11875500BT, Gibco/Lifetechnologies, USA).
Three‐colour pre‐stained protein marker 10 kDa~250 kDa (WJ103, Epizyme, China), OPTI MEM I (31985062, Gibco, USA).
Four‐colour multi‐labeled immunofluorescence staining kit (abs50012, Absin, China).
4× protein loading buffer (containing β‐mercaptoethanol) (P1016, Soleibao, China) Trypsin‐EDTA digestion solution (PBS) (KGY0012, Nanjing KGI, China).
PAGE gel rapid preparation kit 12.5% (PG113, Epizyme, China).
RIPA lysis solution (strong) (PS0013, Leagene, China).
Goat anti‐rabbit TRITC fluorescent secondary antibody kit (BD5005, Bioworld, USA).
Goat anti‐mouse TRITC fluorescent secondary antibody kit (BD5004, Bioworld, USA).
DPP‐IV/CD26 (High‐purity dimer) protein (9168‐SE‐010, R&D systems, USA).
Lipofectamine™ 2000 transfection reagent (11668019, ThermoFisher, USA).
Dynabeads™ protein A for immunoprecipitation (10001D, ThermoFisher, USA) Fluorescent mounting tablets (including DAPI) (ZLI‐9557, Zhongshan Golden Bridge, China).
Western primary antibody diluent (ZS402‐1A, ZOMANBIO, China).
4% tissue cell fixative (P1110, Soleibao, China).
Foetal bovine serum (South America) (MP20002‐500 ml, Yuanye Biology, China).
RPMI 1640 medium (C11875500BT, Gibco/Lifetechnologies, USA).
Three‐colour pre‐stained protein marker 10 kDa~250 kDa (WJ103, Epizyme, China), OPTI MEM I (31985062, Gibco, USA).
Four‐colour multi‐labeled immunofluorescence staining kit (abs50012, Absin, China).
4× protein loading buffer (containing β‐mercaptoethanol) (P1016, Soleibao, China) Trypsin‐EDTA digestion solution (PBS) (KGY0012, Nanjing KGI, China).
PAGE gel rapid preparation kit 12.5% (PG113, Epizyme, China).
RIPA lysis solution (strong) (PS0013, Leagene, China).
3
STAT1 Immunoprecipitation and Analysis
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Cells were lysed in IP lysis buffer (Thermo Scientific; 87787) when their confluency reached 80%. Cell lysate was incubated with either STAT1 antibody (4 μg, Proteintech; 10144-2-AP) or isotype control IgG and Dynabeads™ Protein A for Immunoprecipitation (Thermofisher; 10001D) with rotation overnight at 4 °C, followed by washing three times with PBS/Tween 20 (0.02%), using a magnet to collect the beads after each wash. Fifteen per cent of the precipitated protein sample was subjected to SDS–PAGE. Visualisation was carried out using the Gel Doc™ XR+ Gel Documentation System (Bio-Rad).
4
Proteomic Analysis of Oxidized Cysteine
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