For 35SγGTP incorporation assays, 20 μg of 4xFlag-Rab33b was loaded with unlabeled GDP (5 mM) before ubiquitination as described 22 (link). GDP loaded 4xFlag-Rab33b was used for ubiquitination assays in the presence of either SdeA (10 μg) or SdeAE/A (10 μg) for 2 h at 37°C. 20% of the samples were withdrawn to test for the extent of ubiquitination of 4xFlag-Rab33b by SDS-PAGE and Coomassie staining. Ubiquitinated or non-ubiquitinated 4xFlag-Rab33b was incubated in 50 μL nucleotide exchange buffer containing 25 mM Tris·HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, and 0.1 mM EDTA with 5 μCi 35SγGTP (Perkin-Elmer). GTP-loading reactions were performed at 22°C. Aliquots of reactions were withdrawn at indicated time points, passed through nitrocellulose membrane filters (Hawp02500; Millipore) and placed onto a vacuum platform attached to a waste liquid container. Membranes were washed three times using the exchange buffer to remove the free nucleotides, and were then transferred into scintillation vials containing 8 mL scintillation fluid (Beckman). Incorporated 35SγGTP was detected by a scintillation counter at 1 min per count.
Scintillation fluid
Manufactured by Beckman Coulter
Scintillation fluid is a specialized liquid used in scintillation counting, a technique employed in various scientific and analytical applications. Its primary function is to convert the energy released by radioactive decay or other ionizing events into flashes of light, which can then be detected and quantified by a scintillation counter.
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Lab products found in correlation
3 protocols using scintillation fluid
1
Rab33b Ubiquitination and GTP Incorporation
2
Rab1 GTP loading dynamics
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GST-Rab1 was overexpressed in E. coli together with His6-SetA or His6-SetAD134,136A. Themodification ratios of affinity purified Rab1 were analyzed via mass spectrometry before testing the ability of each to load 35SγGTP (a non-hydrolyzable GTP analog). Nucleotide-free modified and unmodified GST-Rab1 (6.6 µM) were incubated in 100 μL nucleotide exchange buffer containing 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, and 0.1 mM EDTA with 5 mM unlabeled GDP for 2 h at room temperature. 15 μCi 35SγGTP (Perkin-Elmer) in 50 μL nucleotide exchange buffer was added to the samples. His6-SidM (5 μg) was added to indicated reactions to catalyze the loading of radiolabeled GTP analog. Reaction aliquots were withdrawn at indicated time points, placed onto nitrocellulose membrane filters (VSWP02500; Millipore) atop a vacuum platform attached to a waste liquid container. Membranes were washed three times using nucleotide exchange buffer to remove the free nucleotides, and were then transferred into scintillation vials containing 8 mL scintillation fluid (Beckman). Incorporated 35SγGTP was measured by a scintillation counter at 1 min per count.
3
Rab33b Ubiquitination and GTP Incorporation
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For 35SγGTP incorporation assays, 20 μg of 4xFlag-Rab33b was loaded with unlabeled GDP (5 mM) before ubiquitination as described 22 (link). GDP loaded 4xFlag-Rab33b was used for ubiquitination assays in the presence of either SdeA (10 μg) or SdeAE/A (10 μg) for 2 h at 37°C. 20% of the samples were withdrawn to test for the extent of ubiquitination of 4xFlag-Rab33b by SDS-PAGE and Coomassie staining. Ubiquitinated or non-ubiquitinated 4xFlag-Rab33b was incubated in 50 μL nucleotide exchange buffer containing 25 mM Tris·HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, and 0.1 mM EDTA with 5 μCi 35SγGTP (Perkin-Elmer). GTP-loading reactions were performed at 22°C. Aliquots of reactions were withdrawn at indicated time points, passed through nitrocellulose membrane filters (Hawp02500; Millipore) and placed onto a vacuum platform attached to a waste liquid container. Membranes were washed three times using the exchange buffer to remove the free nucleotides, and were then transferred into scintillation vials containing 8 mL scintillation fluid (Beckman). Incorporated 35SγGTP was detected by a scintillation counter at 1 min per count.
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