Recombinant proteins rHis-SseB and rGST-SseB were analyzed by western blotting using the monoclonal anti-His tag (Sigma, Saint Louis, MO, USA), GST mouse mAb (Cell Signaling Technology, Beverly, MA, USA), or antisera against Salmonella Enteritidis C50041 prepared by orally infecting mice with Salmonella Enteritidis C50041. Western blotting was performed as described previously [19 (link)]. Briefly, the contents of the recombinant proteins were separated by SDS-PAGE and electrotransferred to a nitrocellulose membrane. The membranes were probed with a monoclonal anti-His tag (1:3000), anti-GST tag mouse mAb (1:3000), or antisera against Salmonella Enteritidis C50041 (1: 1000) as the primary antibody and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) antibody (1:5000 dilution; Sigma). Immunoreactivity was measured using Super Signal West Pico Chemiluminescent Substrate (Pierce, Chemical, Rockford, IL, USA) according to the manufacturer’s instructions.
Gst mouse mab
Manufactured by Cell Signaling Technology
Sourced in United States
The GST mouse mAb is a monoclonal antibody that specifically recognizes the Glutathione S-Transferase (GST) protein. This antibody can be used for the detection and purification of GST-tagged recombinant proteins in various applications.
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2 protocols using gst mouse mab
1
Recombinant Protein Analysis by Western Blot
2
Clathrin Binding Assay with βArr1 and βArr2
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To measure clathrin binding to βarr1 and βarr2, in the absence or presence of 5:1 (ligand:βarr) molar ratio of ligands (Mdm2ABR peptide or heparin), 20 μg of each βarrs were incubated in a buffer containing 20 mM HEPES pH 8.0, 200 mM NaCl, and 5 mM β-mercaptoethanol at 4 °C for 1 h. After incubation, the volume of each mixture was brought up to 100 μL with the buffer, then the mixture was added 50 μL GST beads (containing 30 μg GST-clathrin) and agitated for 2 h at 4 °C. The GST-clathrin-bound GST beads were then centrifuged at 20,000 × g in a benchtop microcentrifuge, washed with 1 mL of binding buffer five times, and incubated with 100 μL of 15 mM reduced glutathione buffer. Clathrin binding to βarrs was measured by western blot analysis with an anti-his tag mouse mAb (1:5,000, MBL). Secondary antibodies used were anti-mouse IgG, HRP-linked Antibody (Cell Signaling Technology). Blots were reprobed with a GST mouse mAb (1:5,000, Cell Signaling Technology) to ensure equal loading of GST-clathrin for each reaction.
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