GST-Rab5A (Canis lupus) was expressed in BL21 Star (DE3) bacterial cells (#C6010-03, Life Technologies, USA). Protein was purified with glutathione beads (Thermo Scientific; Rockford, IL), analyzed by SDS-PAGE and Coomassie Blue staining, and used for Rab5A pulldown experiments. PI3K-C2γ and Vps15 were synthesized using the TNT Quick Coupled Transcripiton/Translation Systems (Promega; Madison, WI) and Expre35s35s, [35S]-Protein Labeling Mix (Perkin Elmer). GST-Rab5A beads were loaded with either GDP or GTPγS as described 55 (link), and incubated with the [35S]-labeled proteins at 4°C for 2h. After six washes with 20mM Tris pH7.4, 25mM NaCl, 5mM MgCl2, 0.1% NP-40, 1mM DTT, 10μM of either GDP or GTPγS proteins bound to the beads were analyzed by SDS-PAGE and autoradiography.
Tnt quick coupled transcripiton translation systems
Manufactured by Promega
The TNT Quick Coupled Transcription/Translation Systems are in vitro assays that allow for the coupled transcription and translation of proteins from DNA templates. The system provides a convenient and efficient method for the production of proteins in a single reaction.
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Lab products found in correlation
2 protocols using tnt quick coupled transcripiton translation systems
1
Rab5A Pulldown Assay with Effectors
2
Rab5A-mediated Protein Interactions
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GST-Rab5A (Canis lupus) was expressed in BL21 Star (DE3) bacterial cells (#C6010-03, Life Technologies). Protein was purified with glutathione beads (Thermo Scientific), analysed by SDS–PAGE and Coomassie Blue staining, and used for Rab5A pull-down experiments. PI3K-C2γ and Vps15 were synthesized using the TNT Quick Coupled Transcripiton/Translation Systems (Promega) and Expre35s35s, [35S]-Protein Labeling Mix (Perkin Elmer). GST-Rab5A beads were loaded with either GDP or GTPγS as described56 (link), and incubated with the [35S]-labelled proteins at 4 oC for 2 h. After six washes with 20 mM Tris (pH 7.4), 25 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 1 mM dithiothreitol, 10 μM of either GDP or GTPγS proteins bound to the beads were analysed by SDS–PAGE and autoradiography.
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