NanoLuc® (Nluc) Luciferase Antibody

Detection of NanoLuc® Luciferase by Western Blot. Western blot shows Recombinant NanoLuc®. PVDF membrane was probed with 2 µg/mL of Mouse Anti-Human NanoLuc® (Nluc) Luciferase Monoclonal Antibody (Catalog # MAB100261) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for NanoLuc® at approximately 20 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of NanoLuc® (Nluc) Luciferase by Western Blot Luciferase assay for quantifying the release of GPC4 from astrocytes. A, Nluc is inserted at the N terminus, after the endogenous signal peptide, to preserve GPC4 trafficking. B, Primary astrocytes were nucleofected with Nluc-GPC4 and treated with and without PI-PLC. Western botting with alpha -Nluc antibody showed the expected 20 kDa size shift in the N-terminal fragment. PI-PLC treatment facilitates the release of Nluc-GPC4, confirming the GPI-anchorage of the construct. C, Representative trace of one experiment showing the linear kinetics of GPC4 release from astrocyte culture (R2 = 0.998). Error bars indicate the standard error of the mean. D, Astrocytes expressing Nluc-GPC4 were incubated in fresh media with and without PI-PLC for 3 h, and Nluc signal was measured in the cell lysate and media. Nluc signal was normalized to untreated lysate conditions for each biological replicate. PI-PLC treatment resulted in the decrease in the luciferase activity of the cell lysate and the corresponding increase in the activity in the media. These data show Nluc-GPC4 is quantitative in measuring released versus surface pools of GPC4. Error bars indicate 95% CI of the mean here and in following graphs. The requirement of the GPI-anchorage for PI-PLC-dependent release of GPC4 is shown in Extended Data Figure 2-1. E, The release rate (media over lysate activity) of Nluc-GPC4 and Nluc-Prion was normalized to Nluc-GPC4 release. Nluc-GPC4 is released ∼2-fold more than Nluc-Prion (t test p < 0.0001, Cohen’s d = 3.59); ***p < 0.001. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34301723), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of NanoLuc® (Nluc) Luciferase by Western Blot Iterative optimization of the SEPLuc and Antares bioluminescence signal ratios. (A) cyOFP1 was inserted into the existing membrane-anchored SEPLuc fusion via the 5-amino acid linker (n = 2). (B) cyOFP1 was fused to another Nanoluc sequence, then linked downstream of SEPLuc via E2A (n = 2). (C) Antares was cloned downstream of SEPLuc and IRES (n = 2). (D) The Antares-SEPLuc construct. Antares is followed by T2A and puromycin (colored purple), then by IRES and SEPLuc. (n = 3). (E) Comparison of the Antares-SEPLuc constructs utilizing wildtype IRES and the mutant variants IRESv11 and IRESv24 (n = 3). (F) Comparison of the Antares-SEPLuc IRESv24 constructs utilizing two weaker Nanoluc precursors, C1A4E and C1A4E+6, transiently expressed in SW982 cells (n = 2). (G) Western blot of whole cell lysates from SW982 transfected with vector control, Nanoluc, SEPLuc, Antares, Antares-SEPLuc, Antares-SEPLuc IRESv24, and pHLuc. All constructs were transfected into a model cell line, HEK293T for initial assessment of their expression, unless stated otherwise. Downward arrows on spectral scan values indicate the wavelengths at which peaks are expected (450 nm for Nanoluc, 510 nm for SEP, and 580 nm for Antares). Luciferase assay values were normalized to the Nanoluc emission at 450 nm. Error bars are SD. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/32457886), licensed under a CC-BY license. Not internally tested by R&D Systems.