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Luciferase assay

LH Lihui Han
dm dapeng ma

Last updated date: Apr 7, 2021 Views: 859 Forks: 0

original An abbreviated version of this protocol was published in Science Advances in Mar, 2021
Arginine methyltransferase PRMT5 negatively regulates cGAS-mediated antiviral immune response
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Protocols

Luciferase assay:

Luciferase assay is performed with Dual-Luciferase Reporter Assay System kit (Promega, E1910), detailed procedures are as follows:

  1. Plate HEK293T cells into 48-well plate.
  2. Transfect pRL-IFNβ-Luc/pRL-TK or pRL-ISRE-Luc/pRL-TK together with ISD, cGAS, STING and increased amounts of PRMT5 plasmid into HEK293T cells with lipo2000, continue to culture for 24h.
  3. Remove growth medium from the cultured cells, add 65μl 1× Passive lysis buffer (PLB) to collected wells and rock the culture plates at RT for 15min.
  4. Transfer the lysate to a new tube containing 100μl Luciferase Assay Reagent II (LAR II), mix by pipetting 2 or 3 times.
  5. Measure firefly luciferase luminescence.
  6. Add 100µl of Stop & Glo® Reagent.
  7. Measure apparent luminescence.
  8. Subtract background contributed from coelenterazine autoluminescence plus instrument background.
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Han, L and ma, d(2021). Luciferase assay. Bio-protocol Preprint. bio-protocol.org/prep998.
  2. Ma, D., Yang, M., Wang, Q., Sun, C., Shi, H., Jing, W., Bi, Y., Shen, X., Ma, X., Qin, Z., Lin, Y., Zhu, L., Zhao, Y., Cheng, Y. and Han, L.(2021). Arginine methyltransferase PRMT5 negatively regulates cGAS-mediated antiviral immune response. Science Advances 7(13). DOI: 10.1126/sciadv.abc1834

Category

Immunology

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