Lgbit

Manufactured by Promega

Sourced in United States

LgBit is a DNA size selection and purification product for use in molecular biology applications. It is designed to isolate DNA fragments within a specified size range from complex samples.

Automatically generated - may contain errors

Lab products found in correlation

Smbit, by Promega (2 mentions) Quickchange lightning site direct mutagenesis kit, by Agilent Technologies (2 mentions) Lytic buffer, by Promega (2 mentions) Uplsapo 20xo, by Olympus (1 mentions) Furimazine, by Promega (1 mentions) Imagem, by Hamamatsu Photonics (1 mentions) Sb203580, by Merck Group (1 mentions) Brefeldin a, by BioLegend (1 mentions) Pd153035, by Selleck Chemicals (1 mentions) Colchicine, by Fujifilm (1 mentions) Thapsigargin, by Fujifilm (1 mentions) Vincristine, by Fujifilm (1 mentions) Ml240, by Merck Group (1 mentions) Lsm 780, by Zeiss (1 mentions) Pfastbac vector, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

LgBiT-substrate cocktail (4 citations) PBit1.1-C[TK/LgBiT], (4 citations) LgBiT subunit (3 citations) LgBiT protein (3 citations) PCMV-LgBiT expression plasmid (3 citations) LgBiT-β-arrestin 2 (2 citations) Purified LgBiT (2 citations) LgBiT stock solution (2 citations) HEK-293 CCK1R-SmBiT LgBiT-ARRB2 (2 citations) LgBit expression vector (2 citations)

7 protocols using lgbit

Bioluminescent GLUT4 Translocation Assay

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Transfected HEK293T cells were incubated with DMEM containing NLucN (50 nM) and NLuc-substrate furimazine (1:1000, Promega Corp.). For measurement with NlucC-GLUT4, 3T3-L1 cells were serum-starved for 2 h at 37°C in low serum medium (DMEM supplemented with 0.5% FBS).
The cell medium was exchanged to DMEM containing LgBit (1:100, Promega Corp.) and furimazine (1:1000). After insulin stimulation, the cells were incubated for 30 min at 37°C. Luminescence was measured with a plate reader (TriStar930; Berthold Technologies), with a counting time of 1 s.
For luminescence imaging, the cell medium was exchanged to DMEM containing LgBit (1:400, Promega Corp.) and furimazine (1:200). After insulin stimulation, the cells were observed using an inverted fluorescence microscope (IX-81; Olympus Corp.) with a 20× objective lens (UPLSAPO20XO; Olympus Corp.). Luminescence images were acquired with a CCD camera (ImagEM; Hamamatsu Photonics) cooled to -70°C.

Endo M., Miyasaki M., Li Q., Kawamura G, & Ozawa T. (2019). A Detection Method for GLUT4 Exocytosis Based on Spontaneous Split Luciferase Complementation. Analytical sciences : the international journal of the Japan Society for Analytical Chemistry, 35(8).

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Tau Protein Biosensor Construction

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Plasmids encoding for NanoBiT system (SmBit and LgBit) were purchased from Promega (Madison, USA) and cloned upstream of the tau protein in pcDNA3.1 vectors by restriction enzymes. The tau constructs used in this study comprise: (i). tau full-length (aa 1–441 of 2N4R tau); (ii). tau(P301L) (tau full-length displaying the P301L mutation); (iii). K18(P301L) (aa 244–369 comprising the four repeat domains of tau with the P301L mutation). The tau P301L mutants were generated by PCR using QuickChange Lightning Site-Direct mutagenesis kit (Agilent Technologies, Santa Clara, USA). For all intermolecular sensors, we generated N- and C-terminal LgBit/SmBit conjugated tau constructs. All the combination pairs were tested and the best configuration for each biosensor was chosen based on the criteria of displaying a measurable basal signal. Similarly, LgBit and SmBit at either N- or C-terminal positions were also tested for all intramolecular sensors. All constructs include an HA-tag and their sequences were verified by DNA sequencing.

Cecon E., Oishi A., Luka M., Ndiaye-Lobry D., François A., Lescuyer M., Panayi F., Dam J., Machado P, & Jockers R. (2023). Novel repertoire of tau biosensors to monitor pathological tau transformation and seeding activity in living cells. eLife, 12, e78360.

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High-Throughput Screening of LOPAC Library

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At first, 4×10⁴ PC9-KI cells were seeded on 96 well plates. Then, after 24 h, 1μM of compounds in the LOPAC chemical compound library (Sigma Aldrich, MO, USA) were added to each well. Following 24h, the medium was discarded, and a detection mix (PBS 12.5 μL, lytic buffer 12.5 μL, substrate (Promega) 0.25 μL, and LgBiT (Promega) 0.125μL) was added to each well. The plates were incubated at room temperature for 10 min in the dark, and the luminescence was measured using ARVO X3.

Uchida Y., Matsushima T., Kurimoto R., Chiba T., Inutani Y, & Asahara H. (2021). Identification of chemical compounds regulating PD-L1 by introducing HiBiT-tagged cells. FEBS letters, 595(5), 563-576.

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Cytotoxicity Screening of Drug Candidates

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First, 4×10⁴ of PC9-KI cells were seeded on 96 well plates. After 24 h, 0.1 % of DMSO or 10⁻⁵μM, 10⁻⁴μM, 10⁻³μM, 10⁻²μM, 10⁻¹μM, 1 μM, or 10 μM of SB203580 (Sigma Aldrich), brefeldin A (BioLegend, CA, USA), PD153035 (Selleck, TX, USA), colchicine (WAKO), vincristine (Cayman, Michigan, USA), thapsigargin (WAKO, Osaka, Japan), and ML-240 (Sigma Aldrich, MO, USA) were added. After 24 h, the medium was discarded, and detection mix (PBS 12.5 μL, lytic buffer (Promega) 12.5 μL, substrate (Promega) 0.25 μL, and LgBiT (Promega) 0.125 μL) was added to each well.

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Cell-Cell Interaction Assay Protocol

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A culture insert (Ibidi) was placed on the glass part of a glass base 35 mm dish, and then the ligand cells and receptor cells (4 × 10⁴ cells each) were seeded separately in the two wells of the culture insert. After 2 h incubation, the culture insert was removed, and the cells were cultured with 2 ml medium. After 2–3 days incubation, the medium was replaced with the 2 ml fresh medium containing 20 mM HEPES, 1 μl HiBiT substrate (Nano-Glo^® Live Cell EX-4377, Promega) and 8 μl LgBiT (Promega), and the luminescence and fluorescence were imaged with LCV110. Cells labeled with mCherry-CAAX were imaged with a confocal microscope LSM 780 (Carl Zeiss) in Supplementary Fig. 6.

Sekine R., Shibata T, & Ebisuya M. (2018). Synthetic mammalian pattern formation driven by differential diffusivity of Nodal and Lefty. Nature Communications, 9, 5456.

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Cloning and Expression of MC1R and Gαs Constructs

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The full-length human MC1R was cloned into pFastBac vector (Invitrogen) with its native signal peptide replaced by the haemagglutinin (HA) signal peptide followed by a 10 × His tag and cytochrome b562RIL (BRIL) as a fusion partner. To facilitate expression and purification, the C terminus of MC1R was fused with a 15-amino-acid polypeptide linker (GSSGGGGSGGGGSSG) and a LgBiT (Promega).
Human Gαs was constructed based on miniGs (PDB: 5G53) deleting switch III and including eight mutations (G49D, E50N, L63Y, A249D, S252D, L272D, I372A and V375I) 58 (link) . Two additional dominant-negative mutations (G226A and A366S) were introduced to Gαs to decrease the affinity of nucleotide binding and increase the stability of the heterotrimeric G protein 59 (link) . The N terminus (M1-K25) and α-helical domain (AHD, G67-L203) of Gαs were replaced by the N terminus (M1-M18) and AHD (G60-K180) of human Gαi, which was initially designed to bind scFv16 and Fab_G50 60, (link)61 (link) .
Human Gβ with a C-terminal 15-amino-acid polypeptide linker followed by a HiBiT (peptide 86, Promega) and human Gγ were cloned into pFastBac vector, respectively 62 (link) . scFv16 was constructed into the same vector with an N-terminal GP67 signaling peptide.

Ma S., Chen Y., Dai A., Yin W., Guo J., Yang D., Zhou F., Jiang Y., Wang M., & Xu H.E. (2021). Structural mechanism of calcium-mediated hormone recognition and Gβ interaction by the human melanocortin-1 receptor.

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NanoBiT-based Tau Protein Sensors

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Plasmids encoding for NanoBiT system (SmBit and LgBit) were purchased from Promega (Madison, USA) and cloned in front of the tau protein in pcDNA3.1 vectors by restriction enzymes. The tau constructs used in this study comprises: i. tau full-length (aa 1-441 of 2N4R tau); ii. tau(P301L) (tau full-length displaying the P301L mutation);
iii. K18(P301L) (aa 244-39 comprising the 4 repeat domains of tau with the P301L mutation). The tau P301L mutants were generated by PCR using QuickChange Lightning Site-Direct mutagenesis kit (Agilent Technologies, Santa Clara, USA). For all intermolecular sensors, we generated N-and C-terminal LgBit / SmBit conjugated tau constructs. All the combination pairs were tested and the best configuration for each biosensor was chosen based on the criteria of displaying a measurable basal signal.
Similarly, LgBit and SmBit at either N-or C-terminal positions was also tested for all intramolecular sensors. All constructs include a HA-tag and their sequences were verified by DNA sequencing.

Cecon E., Oishi A., Luka M., Ndiaye‐Lobry D., François A., Lescuyer M., Panayi F., Dam J., Machado P., & Jockers R. (2022). Novel repertoire of tau biosensors to monitor pathological tau transformation and seeding activity in living cells.

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