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Nanoglow substrate

Manufactured by Promega

Nanoglow substrate is a reagent used in luminescent assays. It is designed to produce a bright glow when combined with a specific enzyme or protein. The substrate can be used to detect and measure the presence of the target analyte in a sample.

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3 protocols using nanoglow substrate

1

In Vitro and Eukaryotic Expression of ATP4A and ATP4B

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Rluc-ATP4A was expressed in vitro using the TnT SP6 Quick Coupled Transcription/Translation kit (Promega), based on transcription by the SP6 phage RNA polymerase and translation by a rabbit reticulocyte lysate cell-free expression system. Nluc-ATP4B was expressed in eukaryotic cells, using the Expi293 expression system (Thermo Fisher Scientific, Waltham, MA, USA). In the Expi293 expression system, recombinant protein expression is achieved by high efficiency transfection of Expi293F, a derivative of HEK293 cells, adapted to growth in suspension in a defined composition, serum free medium. After 48 h of growth with agitation, transfected Expi293F cells were pelleted and lysed with passive lysis buffer (Thermo Fisher Scientific). Expression of recombinant antigens was assessed by quantification of luciferase activity in the lysates after the addition of Renilla luciferase assay system substrate or NanoGlow substrate (Promega), reconstituted according to the manufacturer instructions, for ATP4A and ATP4B, as appropriate. Luciferase activity was measured using a Berthold Centro xS960 luminometer (Berthold, Germany) and expressed in light units (LU) emitted over a time interval of 2 s. Recombinant antigen preparations were aliquoted and stored frozen at −80 °C.
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2

Rapid HPV-associated HNSCC Diagnosis

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The LIPSTICKS technology [24 (link)] employed cell extract containing NanoLuc-E6 protein was investigated for ultrarapid diagnosis of HPV-associated HNSCC. Compared to the previously reported protocol, a slightly modified version of the assay was used which changed the order of reagent addition and produced a higher signal. To perform the modified version of the assay, 5 μL of the diluted serum sample (1:50 in water) is added to 5 μL of the NanoLuc-E6 fusion protein cell extract (30 million LU/ μL) in a 1.5 mL microfuge tube and then 5 μL of diluted paramagnetic beads (Thermo Scientific/Pierce® protein A/G magnetic beads, Waltham, MA, USA), diluted 1:5 in water are added. The reaction mix is tapped two times to disperse the magnetic beads and then 100 μL of buffer A is pipetted into the reaction mixture and the tube is immediately vortexed for 2 s. A 1/8 diameter neodymium magnetic stick (K & G Magnets, Pipersville, PA, USA) is then immersed into the tube containing the beads for 5 s to collect the immune complexes. The magnet is removed and dipped twice in wash buffer A. Lastly, the magnetic stick is placed in a tube, preloaded with 100 μL of the Nanoglow substrate (Promega) and the luminescent glow is measured with the tube luminometer with an integration time of 1 s.
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3

Rapid Luminescent Antigen Detection

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The portable, handheld EnSURE photodiode luminometer (Hygiena) was also employed for testing. In order configure the device for LIPSTICKS, the Ultrasnap cap tubes supplied by the manufacturer (Hygiena) were first emptied of their contents and then rinsed four times with distilled water. The cleaned, empty tubes were then refilled with 100 μl Nanoglow substrate (Promega). To quantify the light emitted by the antigen/antibody complex, magnets bound with immune complexes were simply dropped into the Ultrasnap tube containing Nanoglow substrate, recapped and placed in the EnSURE luminometer and measured with an integration time of 15 s. Unlike Renilla luciferase antigen fusions, the NanoLuc reporter produced a high output with a stable glow with its substrate and is highly detectable during the long integration time with the handheld luminometer. Due to additional integration time of 15 s needed for the handheld luminometer, these tests required 1 min for completion.
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