GST pull-down assays were performed as previously described19 (link). GST-fused CACUL1 was expressed in Escherichia coli and purified on glutathione Sepharose® beads (GE Healthcare, Chicago, IL, USA). FLAG-SIRT1 or Myc-PPARγ protein was translated in vitro using the TNT® Rabbit Reticulocyte System (Promega). Then, approximately equal amounts of GST or GST-CACUL1 were incubated with in vitro-translated FLAG-SIRT1 or Myc-PPARγ protein. Bound proteins were detected by WB using anti-FLAG or anti-Myc antibodies.
Tnt rabbit reticulocyte system
Manufactured by Promega
The TNT rabbit reticulocyte system is a cell-free in vitro transcription and translation system derived from rabbit reticulocytes. It is designed for the efficient expression of recombinant proteins from DNA templates.
Automatically generated - may contain errors
4 protocols using tnt rabbit reticulocyte system
1
GST Pull-Down Assays for Protein-Protein Interactions
2
Genome-wide DNA Affinity Profiling
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DAP‐seq samples were performed using the TNT rabbit reticulocyte system (Promega) as previously described using 1 μg of genomic B73 DNA library (Bartlett et al., 2017 ; Galli et al., 2018 ). Sequenced reads were mapped to the B73v3 genome. Peaks were called using Gem v.2.5 using a GST‐HALO negative control sample for background subtraction and an FDR of 0.00001 (‐‐q 5). Peaks were associated with their closest putative target genes using chipseeker (Yu et al., 2015 ).
3
GST Pull-Down Assay for SIRT1 Interactions
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GST pull-down assays were conducted as previously described32 (link). Briefly, GST-fused AROS and Cdh1 proteins were expressed in Escherichia coli and refined with glutathione-Sepharose beads (GE Healthcare). Flag-SIRT1 protein was translated in vitro using the TNT rabbit reticulocyte system (Promega). GST protein (2 μg) was incubated with 10 μl of SIRT1 protein. The bound proteins were visualized by WB using an anti-Flag M2 antibody (Sigma‒Aldrich, F1804).
4
Asxl1-HCF-1 Interaction Assay
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Glutathione S-transferase (GST) pull-down assay was performed as described previously3 (link). GST-fused Asxl1 (aa1193–1514) protein was expressed in E. coli and purified using glutathione-Sepharose beads (GE Healthcare). Flag-HCF-1 (aa1–434) protein was translated in vitro using a TNT® rabbit reticulocyte system (Promega). Briefly, 2 μg of GST-Asxl1 (or GST) was incubated with 10μL of Flag-HCF-1 protein. Bound protein was visualized by WB using an anti-Flag M2 monoclonal antibody (Sigma, F-3165).
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