To study if Diaph1 bound to Rab5a or TβRII and TβRII bound to Rab5a in vitro, GST pull down assays were performed.1 (link) GST pull down assays were setup by mixing a detagged protein with 2nd protein conjugated to Glutathione Sepharose 4B beads (molar ratio 2:1) in a binding buffer (25 mM Tris-HCl pH7.6, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1% NP-40, 0.1 mM PMSF, protease inhibitors) in a reaction volume of 200 μL. Protein binding was allowed by incubating the mixture at 10°C for 4 hours followed by centrifugation at 500 ×g for 5 minutes to precipitate the Glutathione Sepharose 4B beads. After washing the beads two times with binding buffer, the protein pulled-down was eluted by Laemmli sample buffer and loaded onto an SDS-PAGE gel for WB. Anti-Rab5 (D-11) (46692; Santa Cruz technology) and anti-TβRII (184948; Abcam) were used to detect the pulled-down proteins. Last, the blots were subjected to Ponceau S staining to visualize the GST-fused proteins used for pull down assays.
Anti rab5 d 11
Manufactured by Santa Cruz Biotechnology
Anti-Rab5 (D-11) is a monoclonal antibody that specifically recognizes the Rab5 protein. Rab5 is a small GTPase that plays a crucial role in the regulation of early endosome formation and vesicle trafficking within cells. The Anti-Rab5 (D-11) antibody can be used to detect and study the localization and expression of Rab5 in various experimental systems.
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2 protocols using anti rab5 d 11
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Diaph1 Binding Interactions Probed
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Diaph1 Binding Interactions Probed
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To study if Diaph1 bound to Rab5a or TβRII and TβRII bound to Rab5a in vitro, GST pull down assays were performed.1 (link) GST pull down assays were setup by mixing a detagged protein with 2nd protein conjugated to Glutathione Sepharose 4B beads (molar ratio 2:1) in a binding buffer (25 mM Tris-HCl pH7.6, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1% NP-40, 0.1 mM PMSF, protease inhibitors) in a reaction volume of 200 μL. Protein binding was allowed by incubating the mixture at 10°C for 4 hours followed by centrifugation at 500 ×g for 5 minutes to precipitate the Glutathione Sepharose 4B beads. After washing the beads two times with binding buffer, the protein pulled-down was eluted by Laemmli sample buffer and loaded onto an SDS-PAGE gel for WB. Anti-Rab5 (D-11) (46692; Santa Cruz technology) and anti-TβRII (184948; Abcam) were used to detect the pulled-down proteins. Last, the blots were subjected to Ponceau S staining to visualize the GST-fused proteins used for pull down assays.
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