In vitro Ub conjugation reactions were performed using the purified His-tagged UBC22 as described above. The Ub thioester/conjugation initiation reagents were purchased from Boston Biochem (K-995). Unless noted otherwise, the reaction mixture contained 5 μM purified His-UBC22 and 62.5 μM Ub in the supplied reaction buffer; concentrations of other components were used following the manufacturer’s instructions. K11R, K48R, and K63R mutant Ub proteins were purchased from Boston Biochem (UM-K11R, UM-K48R, and UM-K63R). The conjugation reactions were performed at 30 °C for 4h. Samples were added with 4X non-reducing loading buffer [200mM Tris-HCl (pH 6.8), 8% SDS, 0.2% bromophenol blue, 40% glycerol] and heated at 95 °C for 5min, then subjected to SDS-PAGE (15% gel). Ub and Ub dimers were detected by western blotting using a monoclonal mouse anti-Ub antibody (P4D1, Cell Signaling Technology) and a horseradish peroxidase-conjugated goat anti-mouse antibody (Bio-Rad). The signal was visualized with ECL Prime reagent (GE Health) according to the manufacturer’s instructions.
Horseradish peroxidase conjugated goat anti mouse antibody
Manufactured by Bio-Rad
Sourced in United States
Horseradish peroxidase-conjugated goat anti-mouse antibody is a secondary antibody that binds to mouse primary antibodies. It is used to detect and quantify target proteins in various immunological techniques, such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ecl prime reagent, by GE Healthcare (1 mentions)
K 995, by R&D Systems (1 mentions)
Um k11r, by R&D Systems (1 mentions)
Um k63r, by R&D Systems (1 mentions)
Biotrace pvdf membrane, by Pall Corporation (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Bio-Rad (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Srebp1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2 resin, by Merck Group (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Anti flag, by Merck Group (1 mentions)
4 protocols using horseradish peroxidase conjugated goat anti mouse antibody
1
In vitro Ub Conjugation Assay
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In vitro Ub conjugation reactions were performed using the purified His-tagged UBC22 as described above. The Ub thioester/conjugation initiation reagents were purchased from Boston Biochem (K-995). Unless noted otherwise, the reaction mixture contained 5 μM purified His-UBC22 and 62.5 μM Ub in the supplied reaction buffer; concentrations of other components were used following the manufacturer’s instructions. K11R, K48R, and K63R mutant Ub proteins were purchased from Boston Biochem (UM-K11R, UM-K48R, and UM-K63R). The conjugation reactions were performed at 30 °C for 4h. Samples were added with 4X non-reducing loading buffer [200mM Tris-HCl (pH 6.8), 8% SDS, 0.2% bromophenol blue, 40% glycerol] and heated at 95 °C for 5min, then subjected to SDS-PAGE (15% gel). Ub and Ub dimers were detected by western blotting using a monoclonal mouse anti-Ub antibody (P4D1, Cell Signaling Technology) and a horseradish peroxidase-conjugated goat anti-mouse antibody (Bio-Rad). The signal was visualized with ECL Prime reagent (GE Health) according to the manufacturer’s instructions.
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In vitro Ub conjugation reactions were performed using the purified His-tagged UBC22 as described above. The Ub thioester/conjugation initiation reagents were purchased from Boston Biochem (K-995). Unless noted otherwise, the reaction mixture contained 5 μM purified His-UBC22 and 62.5 μM Ub in the supplied reaction buffer; concentrations of other components were used following the manufacturer’s instructions. K11R, K48R, and K63R mutant Ub proteins were purchased from Boston Biochem (UM-K11R, UM-K48R, and UM-K63R). The conjugation reactions were performed at 30 °C for 4h. Samples were added with 4X non-reducing loading buffer [200mM Tris-HCl (pH 6.8), 8% SDS, 0.2% bromophenol blue, 40% glycerol] and heated at 95 °C for 5min, then subjected to SDS-PAGE (15% gel). Ub and Ub dimers were detected by western blotting using a monoclonal mouse anti-Ub antibody (P4D1, Cell Signaling Technology) and a horseradish peroxidase-conjugated goat anti-mouse antibody (Bio-Rad). The signal was visualized with ECL Prime reagent (GE Health) according to the manufacturer’s instructions.
2
Western Blot Analysis of ALX3 in B16-F1 Cells
3
Western Blot Analysis of SREBP-1
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SREBP-1 (1:1000) and β-actin (Santa Cruz, Dallas, TX, USA) (1:5000) antibodies were used for western blot assay. Secondary horseradish peroxidase-conjugated goat anti-mouse antibody (Bio-Rad, Hercules, CA, USA) were used at a 1:5000 dilution and detected by the Enhanced Chemiluminescence Reagent (Millipore, Billerica, MA, USA).
4
Co-Immunoprecipitation of SMG6 and UPF1/ILF2
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For co-IP studies, HEK 293T cells (cultured as described above) were co-transfected with plasmids of HA-SMG6 constructs and Flag-His-UPF1fl using polyethyleneimine. The SMG6 constructs were also co-transfected with Flag-ILF2 as a negative control. Transfected cells were incubated at 32°C and harvested 72 h later. Cell extracts were prepared in 250 μl NET-G buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, 10% glycerol); 12 μl of anti-Flag M2 resin (Sigma) were added to 200 μl of each co-transfected cell extract and incubated at 4°C for 1 h. The beads were washed four times with 1 ml NET-G buffer to remove non-specifically bound proteins, while Flag-tagged proteins and their interaction partners were eluted with 40 μl of non-reducing SDS sample buffer. The eluted proteins were resolved by SDS-PAGE and then transferred onto polyvinylidene difluoride membranes for analysis by western blotting. The membranes were probed with mouse monoclonal anti-HA (Covance) and anti-Flag (Sigma) antibodies to detect SMG6 and UPF1/ILF2, respectively. A horseradish peroxidase-conjugated goat anti-mouse antibody (Bio-Rad) used in combination with ECL prime western blotting detection reagent (GE Healthcare) enabled detection of the tagged proteins by chemiluminescence.
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