HEK293 cells were cultured on chamber slides coated with 0.01% collagen type II and transfected with pFLAG-Asc-1 using lipofectamine 2000 (ThermoFisher Scientific, MA, USA) according to the manufacturer’s protocol. At 24 hours after transfection, cells were washed in PBS, fixed in 4% paraformaldehyde PBS at 4 °C for 2 h, and immersed in a blocking buffer [5% normal goat serum, 0.3% TritonX-100 PBS] at 4 °C for 1 h. Then, cells were immunolabeled with a mouse monoclonal antibody to FLAG (1:100, Sigma Aldrich, MO, USA) and a rabbit polyclonal antibody to Asc-1 (1:100) in blocking buffer at 4 °C overnight. The cells were incubated with an FITC-conjugated goat antibody to mouse IgG and a Texas red-conjugated goat antibody to rabbit IgG (both 1:200, Jackson ImmunoResearch Laboratories, PA, USA).
Mouse monoclonal antibody to flag
Manufactured by Merck Group
Sourced in United States
Mouse monoclonal antibody to FLAG is a laboratory reagent used to detect and purify proteins tagged with the FLAG epitope. It is a highly specific antibody that recognizes the FLAG peptide sequence (DYKDDDDK) with high affinity.
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2 protocols using mouse monoclonal antibody to flag
1
Immunofluorescence Assay for FLAG-Asc-1 Expression
2
Transient Transfection and Co-immunoprecipitation in HEK 293T Cells
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Human embryonic kidney (HEK 293T) cells were grown in DMEM supplemented with 10% FBS. Transient transfection and coimmunoprecipitation were performed as described previously (Ganner et al., 2009 (link)). In brief, transfections were performed using the calcium phosphate method; cells were washed with cold PBS and lysed in 1% Triton X-100, 20 mM Tris, pH 7.5, 50 mM NaCl, 50 mM NaF, 15 mM Na4P2O7, 0.1 mM EDTA, and 2 mM Na3VO4 supplemented with protease inhibitor mix (Roche). Lysates were incubated with anti-Flag M2-agarose beads (Sigma-Aldrich) for 2 h and washed with lysis buffer. For Western blots, proteins were fractionated by SDS-PAGE and detected by mouse monoclonal antibody to Flag (Sigma-Aldrich) and GFP (Santa Cruz Biotechnology) and rabbit polyclonal antibody to Flag (Sigma-Aldrich) and V5 (Millipore).
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