In vitro transcription and translation of the NHERF2 and ERα proteins were done using the TNT transcription/translation system (Promega) in the presence of [35S]-methionine. The GST pull-down assays were done by incubating equal amounts of GST, GST-NHERF2 or GST-ERα-domains immobilized on GST beads (Amersham Pharmacia Biotech, Piscataway, NJ) with in vitro-translated recombinant protein. Bound proteins were isolated by incubating the mixture for 3 h at 4°C and then washing five times with NP40 lysis buffer (20 mmol/l Hepes pH 7.9, 100 mmol/l NaCl, 1 mmol/l ethylenediaminetetraacetic acid (EDTA) pH 8.0, 4 mmol/l MgCl2, 1 mmol/l DTT, 0.02% NP40, 10% glycerol and 0.5 mmol/l PMSF). For endogenous ERα pull-down assay, the GST bound proteins were incubated with a whole cell extract of MCF7 cells, resuspended in TBS-0.2% triton X-100 and sonicated. The proteins were eluted with a 2x Laemmli sample buffer, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and visualized by autoradiography or immunoblot.
Tnt transcription translation system
Manufactured by Promega
Sourced in United States
The TNT transcription/translation system is a cell-free, coupled in vitro transcription and translation system that allows for the rapid expression of proteins from DNA templates.
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Lab products found in correlation
3 protocols using tnt transcription translation system
1
NHERF2 and ERα Protein Interaction Assay
2
GST-fusion Protein Interaction Assay
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Plasmid expressing GST fusion proteins or GST control were transformed into E coli BL21, followed by supplemented with 1 mmol/L IPTG to induce protein expression. Bacterial bodies were ultrasonicated at 40% power to harvest the lysate. Glutathione‐Sepharose 4B beads were added to the supernatant, and the mixtures were incubated for 30 minutes at 4°C. The in vitro‐translated proteins were prepared using the TNT transcription/translation system (Promega, USA). The GST fusion proteins were mixed with the in vitro‐transcribed/translated products and incubated in binding buffer for 2 hours at 4°C. The beads were washed five times with binding buffer and denatured in 2 × loading buffer at 95°C for 5 minutes. Protein bands were detected by Western blot using specific antibodies (Table S2 ).
3
In Vitro Co-Immunoprecipitation of PKS1 and GβL
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pGBKT7 and pGADT7, containing the catalytic domain of Pipks1 and full length Pigbl1, respectively, were used as templates for in vitro transcription and translation reactions, using 35S-methionine with the TNT transcription/translation system (Promega). This produced the c-Myc-tagged catalytic domain of PKS1 and HA-tagged GβL. Co-immunoprecipitation was performed using the Matchmaker Co-IP kit (Clontech). The proteins were incubated at room temperature for 1 h, then for 1 h with c-Myc antibody, and then for 1 h with Protein A beads. After pelleting and elution of bound proteins, protein bands were identified by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by phosphorimager analysis.
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