An attR1-ccdB-attR2 cassette (from pBWH) was generated by PCR and cloned into the NotI site of pHTN HaloTag® CMV-neo (Promega) by Gibson assembly to generate pHTNW. An attR1-ccdB-attR2 cassette (from pAWH) was generated by PCR and cloned into the NotI site of pNLF1-N [CMV/Hygro] (Promega) by gibson, to give pNLF1W. Indicated ORFs were cloned into either pHTNW or pNLF1W by Gateway LR reaction (Thermo Fisher Scientific) following the manufacturer’s indications. HEK293T cells were plated in 24-well plates at a density of 1.4 × 105 cells per well. Cells were transfected with 500 ng of pHTNW-ORF, 5 ng of pNLF1W-ORF, 0.75 μl of Lipofectamine 3000 and 1 μl of P3000 Reagent (Thermo Fisher Scientific). After 20 h, each transformation was re-plated in four wells of 96-well plates at a density of 1 × 104 cells per well for duplicate control and experimental samples (technical replicates), and PPIs were analysed with NanoBRET™ Nano-Glo® Detection System kit (Promega) following manufacturer’s instructions. Each transformation experiment was performed at least twice. The corrected NanoBRET ratio was calculated according to the manufacturer’s instructions.
Nanobret nano glo detection system kit
Manufactured by Promega
The NanoBRET™ Nano-Glo® Detection System kit is a laboratory equipment product that enables the detection of nanoBRET signals. It utilizes the Nano-Glo® technology for luminescence-based detection.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Gateway lr reaction, by Thermo Fisher Scientific (2 mentions)
P3000 reagent, by Thermo Fisher Scientific (2 mentions)
Phtn halotag cmv neo, by Promega (1 mentions)
Pnlf1 n cmv hygro, by Promega (1 mentions)
Gateway bp reaction, by Thermo Fisher Scientific (1 mentions)
2 protocols using nanobret nano glo detection system kit
1
Protein-Protein Interaction Analysis via NanoBRET
2
NanoBRET-based Protein-Protein Interactions
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pHTNW (Addgene #136403) and pNLF1W (Addgene #136404) have been previously described (Yang et al. 2018). ORFs of interest were cloned into pENTR vector by Gateway BP reaction and then transferred either to pHTNW or pNLF1W by Gateway LR reaction (Thermo Fisher Scientific) following the manufacturer's protocol. LR reaction were transformed into 10 ul of OmniMAX™2 competent cells (Thermo Fisher Scientific). Positive clones were confirmed by restriction analysis using BsrGI enzyme and by sequencing.
HEK293T cells were plated in 24-well plates at a density of 1.4 × 105 cells per well. Cells were transfected with 500 ng of pHTNW-ORF, 5 ng of pNLF1W-ORF, 0.75 μl of Lipofectamine 3000 and 1 μl of P3000 Reagent (Thermo Fisher Scientific). After 20 h, each transformation was re-plated in four wells of 96-well plates at a density of 1 × 104 cells per well for duplicate control and experimental samples (technical replicates), and PPIs were analyzed with NanoBRET™ Nano-Glo® Detection System kit (Promega) following manufacturer's instructions. Each transformation experiment was performed at least twice. The corrected NanoBRET ratio was calculated according to the manufacturer's instructions.
HEK293T cells were plated in 24-well plates at a density of 1.4 × 105 cells per well. Cells were transfected with 500 ng of pHTNW-ORF, 5 ng of pNLF1W-ORF, 0.75 μl of Lipofectamine 3000 and 1 μl of P3000 Reagent (Thermo Fisher Scientific). After 20 h, each transformation was re-plated in four wells of 96-well plates at a density of 1 × 104 cells per well for duplicate control and experimental samples (technical replicates), and PPIs were analyzed with NanoBRET™ Nano-Glo® Detection System kit (Promega) following manufacturer's instructions. Each transformation experiment was performed at least twice. The corrected NanoBRET ratio was calculated according to the manufacturer's instructions.
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