Agrobacterium strain GV3101 carrying constructs expressing GFP- or Myc-fused proteins were mixed and infiltrated into N. benthamiana leaves as described above. Before cell death was visible (at about 20 h post infiltration), the total leaf proteins were extracted in 0.5 ml plant extraction buffer (CWBIO, Beijing, China), followed by centrifugation at 12 000 g for 20 min. Portions of the supernatants were saved as input samples for later SDS–PAGE and immunoblot analysis. For the Co-IP assay, the remaining supernatant was incubated with 4 μg of anti-Myc monoclonal antibody (Proteintech, Chicago, IL, USA) at 4°C for 2–4 h. The antibody–protein complexes were captured by incubation with 18 μl of agarose resin conjugated with protein A/G for 2–4 h at 4°C. After washing six times with IP buffer, the immunocomplexes were heated at 95°C for 5 min in 50 μl 1× SDS loading buffer. The supernatant after centrifugation was used for immunoblot analysis (Gao et al., 2020 (link)).
Anti myc monoclonal antibody
Manufactured by Proteintech
Sourced in United States, China
The Anti-Myc monoclonal antibody is a laboratory reagent used for the detection and purification of proteins tagged with the c-Myc epitope. It is a mouse-derived monoclonal antibody that specifically binds to the c-Myc tag, a commonly used protein tag for various experimental applications.
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3 protocols using anti myc monoclonal antibody
1
Co-immunoprecipitation of GFP-/Myc-fused Proteins
2
Heterologous Protein Expression Analysis
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After 1 mM IPTG-induced expression overnight, E. coli C41(DE3)/pET28a-Hbp passenger-NGO2105β, E. coli C41(DE3)/pET28a-Hbp passenger and E. coli C41(DE3)/pET28a cells were collected by centrifugation, washed and resuspended in PBS with 1% BSA and 0.01% Tween 20. The anti-Myc monoclonal antibody (1:100) (Proteintech Group, Inc., Wuhan, China) was used as the primary antibody, and FITC-conjugated goat anti-mouse IgG (1:200) (Proteintech Group, Inc., Wuhan, China) was used as the secondary antibody. After three washes with PBS, cells were analyzed by flow cytometry analysis and immunofluorescence microscopy.
3
Immunoblotting with Signaling Inhibitors
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The lysosome inhibitors chloroquine (CQ), NH4Cl, and mouse anti-Flag monoclonal antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). The protein synthesis inhibitor cycloheximide (CHX), the proteasome inhibitor MG132, and rabbit anti-NF-κB p65 and anti-phospho-NF-κB p65 (Ser536) polyclonal antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Mouse anti-β-actin and anti-phospho-IRF3 monoclonal antibodies were obtained from Abcam (Cambridge, UK). Mouse anti-HA polyclonal antibody and anti-Myc monoclonal antibody and rabbit anti-IRF3 and anti-Flag polyclonal antibodies were purchased from Proteintech (Wuhan, China).
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