Tnt t7 quick coupled system

The BB0173 Lep-derived constructs and BB0173 truncated constructs were transcribed and translated using the TNT T7 Quick Coupled System (Promega, Madison, WI). The reactions contained 75 ng of DNA template, 0.5 μl of [³⁵S]Met (5μC_i), and 0.25 μl of microsomes (tRNA Probes) were incubated for 90 min at 30 °C. The translation products were ultracentrifuged (100,000 g for 15 min) on a sucrose cushion, and analyzed by SDS-PAGE. The bands were quantified using a Fuji FLA-3000 phosphoimager and Image Reader 8.1j software.
For the proteinase K protection assay, 2 μl of proteinase K (1 mg/ml) was added to the sample, and the digestion reaction was incubated for 15 min on ice. Before SDS-PAGE analysis, the reaction was stopped by adding 1 mM phenylmethanesulfonyl fluoride (PMSF).
For EndoH (New England Biolabs, Beverly, MA) treatment, 1 μl of 10X Glycoprotein Denaturing Buffer, 1 μl of 10X GlycoBuffer, 1 μl of EndoH and 7 μl of H₂0 were added to make a 10 μl total reaction volume and incubated for 1 h at 37 °C with 0.1 mU of EndoH. The samples were analyzed by SDS-PAGE.

All enzymes, as well as plasmid pGEM1, TNT T7 Quick Coupled System and rabbit reticulocyte lysate were from Promega (Madison, WI, USA). ER rough microsomes from dog pancreas were from tRNA Probes (College Station, TX, USA). EasyTag™ EXPRESS³⁵S Protein Labeling Mix, [³⁵S]-L-methionine and ³⁵S-L-cysteine, for in vitro labeling was purchased from Perkin Elmer (Waltham, MA, USA). Restriction enzymes and Endoglycosidase H were from Roche Molecular Biochemicals (Basel, Switerland). Proteinase K was from Sigma-Aldrich (St Louis, MO). The DNA plasmid, RNA clean-up and PCR purification kits were from Thermo Fisher Scientific (Ulm, Germany). All oligonucleotides were purchased from Macrogen (Seoul, South Korea).

The Lep-derived constructs and truncated constructs were transcribed and translated using the TNT T7 Quick Coupled System (Promega, Madison, WI). The reactions contained 75 ng of DNA template, 0.5 μl of EasyTag (5.5 μCi), and 0.25 μl of microsomes (tRNA Probes, College Station, TX) were incubated for 40 min at 30 °C. The translation products were ultracentrifuged (100,000×g for 15 min) on a sucrose cushion, and analysed by SDS-PAGE. The bands were quantified using a Fuji FLA-3000 phosphoimager and Image Reader 8.1j software.
For the proteinase K protection assay, the translation mixture was supplemented with 1 µL of 50 mM CaCl₂ and 1 μl of proteinase K (2 mg/ml), and the digestion reaction was incubated for 40 min on ice^{47 (link),51 (link)}. Before SDS-PAGE analysis, the reaction was stopped by adding 1 mM phenylmethanesulfonyl fluoride (PMSF). All the translation/glycosylation experiments were repeated at least four times.

The Lep-derived constructs were assayed using the TNT T7 Quick Coupled System (#L1170, Promega). Each reaction containing 1 µL of PCR product, 0.5 µL of EasyTag™ EXPRESS 35S Protein Labeling Mix (Perkin Elmer) (5.5 µCi), and 0.3 µL of microsomes (tRNA Probes) was incubated at 30 °C for 90 min. Samples were analyzed by SDS-PAGE. The bands were quantified using a Fuji FLA-3000 phosphoimager and the Image Reader 8.1 software. Free energy was calculated using: ∆G_app = −RT lnK_app, where K_app = f2g/f1g being f1g and f2g the fraction of single glycosylated and double glycosylated protein, respectively. Endoglycosidase H treatment (Roche) was carried according to the specifications of the manufacturer.

Electrophoretic mobility-shift assay was performed as described5 (link) with modifications as below. Double-stranded oligonucleotides (consensus FXRE/IR1 and SHP/IR1, 5′-GATGGGCCAAGGTCAATGACCTCGGGG-3′ and 5′-CCCTGGTACAGCCTGGGTTAATGACCCTGTTTATGCACTTG-3′, respectively) were end-labelled with [γ-32P] ATP (3,000 Ci per mmol) using T4 polynucleotide kinase for probe labelling. Human FXR and RXRα proteins (Fig. 2c) were prepared with a TNT T7 Quick coupled system (Promega, Madison, WI). In vitro translated proteins were incubated with 0.05 ng of probe in the binding reaction buffer containing 10 mM Tris (pH 8.0), 60 mM KCl, 0.1% NP-40, 6% glycerol, 1 mM dithiothreitol and1 μg of poly(dI-dC) on ice for 20 min. DNA-protein complexes were resolved on standard non-denaturing 4% polyacrylamide gels. The gels were dried and exposed to X-ray film at −80 °C for 5–24 h.