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Luciferase and luciferin

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Luciferase and luciferin are biochemical compounds used in bioluminescence assays. Luciferase is an enzyme that catalyzes a reaction with luciferin, a substrate, resulting in the emission of light. This light production can be used to quantify the presence or activity of the luciferase-luciferin system in various experimental applications.

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Mithras lb 940 multimode plate reader, by Berthold Technologies (1 mentions) Infinite m200 pro, by Tecan (1 mentions) Infinite 200 pro, by Tecan (1 mentions)

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Luciferin

6 protocols using luciferase and luciferin

1

Quantifying ATP Flux in C2C12 Cells

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The ATP flux was examined by luminometry, as previously described5 (link),16 (link). C2C12 cells stably expressing pEF1, pEF1/Panx3, or Ser68Ala were seeded at 1.0 × 104 cells/well in a 96-well plate, and cultured for 2 days in DMEM containing 10% FBS. The cells were then washed with PBS, followed by incubation in PBS for 2 min. The supernatant was collected and assayed with luciferase and luciferin (Promega). The luminescence was measured using a Mithras LB 940 multimode plate reader (Berthold).
Ishikawa M., Williams G., Forcinito P., Ishikawa M., Petrie R.J., Saito K., Fukumoto S, & Yamada Y. (2019). Pannexin 3 ER Ca2+ channel gating is regulated by phosphorylation at the Serine 68 residue in osteoblast differentiation. Scientific Reports, 9, 18759.
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2

Transcriptional Activation Analysis of CBF3

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This experiment was performed as described previously (Qian et al., 2019 (link)). PCR amplified and cloned the ADF5 promoter sequence into pGWB235-LUC to construct the reporter vector. The full-length CBF3 coding sequence was amplified using PCR, and it was cloned into the pBIB-35s-GWR-flag vector as the effect vector. The reporter and effector vectors were co-transformed into N. benthamiana leaves for transcriptional activation analyses. A reporting vector alone was used as a negative control. Fluorescence of the luciferase and luciferin (Promega) reaction was obtained using a Lumazone CA1300B camera (Photometrics). The primers used to clone are listed in Supplementary Table 1.
Zhang P., Qian D., Luo C., Niu Y., Li T., Li C., Xiang Y., Wang X, & Niu Y. (2021). Arabidopsis ADF5 Acts as a Downstream Target Gene of CBFs in Response to Low-Temperature Stress. Frontiers in Cell and Developmental Biology, 9, 635533.
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3

Purification and Characterization of Chaperone Proteins

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Hsp90Ec wild-type and mutants [23 (link)], DnaK wild-type and mutants [55 (link)], CbpA [57 (link)], GrpE [55 (link)] and His-tagged L2 [58 (link)] were isolated as described. All proteins were >95% pure as determined by SDS-PAGE. All DnaK substitution mutants exhibited similar physical properties as the wild-type, including partial proteolysis patterns with and without ATP (Supplemental Fig. S9a) and CD spectra (Supplemental Fig. S9b). All exhibited some DnaK functional activities, including: reactivation of GFP with DnaJ and GrpE (Supplemental Fig. S10a) and further stimulation of GFP reactivation by ClpB (Supplemental Fig. S10b), client binding (L2) (Supplemental Fig. S10c) and basal ATPase activity (Supplemental Fig. S10d). The Hsp90Ec E584C-biotin mutant was similar to Hsp90Ec wild-type in luciferase reactivation activity (Supplemental Fig. S11a). Luciferase and luciferin were from Promega. Concentrations given are for Hsp90Ec, CbpA and GrpE dimers and DnaK, L2 and luciferase monomers. Hsp90Ec E584C was labeled using a 20-fold excess of Maleimide-PEG11-Biotin (Thermo, Life Technologies) as recommended by the manufacturer. CbpA was labeled using a 1.5-fold excess of NHS-PEG4-Biotin (Thermo, Life Technologies). Excess biotin reagent was removed by extensive dialysis.
Kravats A.N., Doyle S.M., Hoskins J.R., Genest O., Doody E, & Wickner S. (2016). Interaction of E. coli Hsp90 with DnaK involves the DnaJ binding region of DnaK. Journal of molecular biology, 429(6), 858-872.
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4

Luciferase Reactivation Assay Protocol

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Luciferase reactivation was performed as previously described [27 (link), 33 (link)]. 20 nM heat-denatured luciferase, prepared as described [30 (link)], was incubated at 24 °C in reaction mixtures (75 μL) containing 25 mM Hepes, pH 7.5, 50 mM KCl, 0.1 mM EDTA, 2 mM DTT, 10 mM MgCl2, 50 μg/ml bovine serum albumin (BSA), 1 mM ATP, an ATP regenerating system (25 mM creatine phosphate, 6 μg creatine kinase), 1 μM Ssa1 wild-type or mutant and 0.025 μM Ydj1 as indicated. Aliquots were removed at the indicated times and light output was measured using a Tecan Infinite M200Pro in luminescence mode with an integration time of 1000 ms following the injection of luciferin (50 μg/ml). Reactivation was determined compared to a non-denatured luciferase control. Luciferase and luciferin were from Promega.
Doyle S.M., Hoskins J.R., Kravats A.N., Heffner A.L., Garikapati S, & Wickner S. (2019). Intermolecular interactions between Hsp90 and Hsp70. Journal of molecular biology, 431(15), 2729-2746.
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5

Purification and Characterization of Chaperone Proteins

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Hsp90Ec wild-type and mutants [30 (link)], DnaK wild-type and mutants [68 (link)], CbpA [69 (link)], Hsp82 wild-type and mutants [27 (link), 33 (link)], Ssa1 wild-type and mutants [67 (link), 70 (link)], Ydj1 [67 (link), 70 (link)], Sis1 [33 (link)], Sti1 [27 (link)] and His-tagged L2 [71 (link)] were isolated as described. All proteins were >95% pure as determined by SDS-PAGE. Luciferase and luciferin were from Promega. Concentrations given are for Hsp90Ec, Hsp82, CbpA, Sis1 and Ydj1 dimers and DnaK, Ssa1, Sti1, GFP, L2 and luciferase monomers. Previously unpublished mutant proteins including DnaK-G328C and D211C, Ssa1-R169H, D172N, E210R, T219C, K322C and L323C, and Hsp82-E402C and E409C were shown to have trypsin digestion patterns and functional activity similar to the wild-type protein (Supplementary Fig. S1a; Supplementary Fig. S2e and f; Supplementary Fig. S3a and b; Supplementary Fig. S6a–d). Hsp90Ec, Hsp82, Ydj1 and Sti1 were labeled with biotin using a 1.5-fold excess of NHS-PEG4-Biotin (Thermo, Life Technologies). Excess biotin reagent was removed using 7K MWCO Zeba Spin Desalting Columns.
Doyle S.M., Hoskins J.R., Kravats A.N., Heffner A.L., Garikapati S, & Wickner S. (2019). Intermolecular interactions between Hsp90 and Hsp70. Journal of molecular biology, 431(15), 2729-2746.
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6

Quantifying Osteogenic ATP Flux

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ATP flux was examined by luminometry, as previously described5 (link),46 (link). Primary calvarial cells isolated from Panx3 KO mice were seeded at 104 cells/well in a 96-well plate, and induced by osteogenic culture media for 14 days. The cells were then washed with PBS, followed by incubation in PBS for 2 min. The supernatant was collected and assayed with luciferase and luciferin (Promega). The luminescence was measured using an Infinite 200 PRO (TECAN).
Tagaya Y., Kurys G., Thies T.A., Losi J.M., Azimi N., Hanover J.A., Bamford R.N, & Waldmann T.A. (1997). Generation of secretable and nonsecretable interleukin 15 isoforms through alternate usage of signal peptides. Proceedings of the National Academy of Sciences of the United States of America, 94(26), 14444-14449.
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