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Successful Luminescent Assays Need Sensitive Instruments

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Abstract

Success in performing bioluminescent assays is about more than just the light signal generated during the experiment. You need an instrument sensitive enough to measure weak luminescence. Some detection instruments are unable to measure samples with low-level signals, meaning you could miss an important "hit" or result. This article covers the topic of signal sensitivity in detection instruments and how you may be losing data points if your instrument cannot measure light accurately.

Promega Corporation

Publication Date: September 2017; tpub_190

Introduction

A wide variety of applications for drug discovery require sensitive, quantitative assays. For example, studying cell signaling pathways to develop and understand new drug therapies require assays that are able to distinguish small changes in transcription, molecular interactions and cellular health. For example, studying transcription can involve characterizing promoters and enhancers of transcription factors, identifying genetic point mutations or deletions, or even cellular stress causing changes in environmental conditions. Knowing how these factors affect pathway outcomes requires the ability to monitor small and subtle differences between experimental and control samples.

Luminescence, fluorescence and absorbance are the three most common methods used in these assays. In general, luminescent assays are more sensitive than fluorescent assays, and fluorescent assays are more sensitive than absorbance assays. Whatever the assay method, detecting low level or small changes in a target requires a sensitive assay. For example, when studying an enzymatic reaction, you don't want to have to add a high level of substrate enzyme or catalyst to drive the reaction to a detectable level because this creates an artificial system that does not reflect physiological conditions. Instead you want your assay to be sensitive enough to be performed at near physiological levels, thus generating more biologically relevant data.


Sensitive Assays Need Sensitive Detection Platforms

Sensitive assays, however, also require a sensitive detection platform. Plate readers that are used to detect luminescence, fluorescence and absorbance signals can vary in sensitivity (Figure 1). Factors that contribute to an instrument’s sensitivity, or limit of detection, include background noise from the detector and electronics, the type of detector that is used, and the instrument’s configuration and overall design. Lowering the instrument’s background improves the limit of detection, which in turn improves the signal-to-noise ratio, providing more usable data from each experiment. Conversely, a high background can over shadow low‐level signals, reducing the usable data from each experiment. To get a true picture of the biology in the cell, it is more relevant to measure your assay at near physiological conditions if possible (Figure 2).

Figure 1. Sensitivity of luminescent detection on GloMax® systems and other commercially available plate readers.

The Bio-Glo™ Luciferase Assay System was used to test instrument sensitivity, and the limit of detection (LOD) was calculated.

The Bio-Glo™ Luciferase Assay System was used to test instrument sensitivity, and the limit of detection (LOD) was calculated.

//embed.widencdn.net/img/promega/0xdvronuy9/640px/14496MA-W.jpeg?u=7fvzhm
Figure 2. Stabilizing HIF1A-HiBiT by incubation with the hypoxia mimetic 1,10-phenanthroline.

HeLa cells were transiently transfected with different amounts of CMV- or PGK-driven expression constructs for HIF1A-HiBiT, diluted in carrier DNA. In parallel, HiBiT was tagged to the endogenous locus in HeLa cells using CRISPR/Cas9, and a clone was isolated. Untransfected cells, transfected cells and the endogenously tagged cell line were plated in 96-well plates and treated the following day for 4 hours with a titration of 1,10-phenanthroline. The Nano-Glo® HiBiT Lytic Reagent was added to all wells, and luminescence was measured after 10 minutes. Panel A. Background-subtracted luminescence shows the varying expression levels of HIF1A-HiBiT in transiently-transfected cells compared to expression from the endogenous promoter. Panel B. Data normalized to untreated cells shows how overexpression of HIF1A-HiBiT reduces the fold response from treatment with 1,10-phenanthroline. Error bars represent the standard deviation for n = 6.

HeLa cells were transiently transfected with different amounts of CMV- or PGK-driven expression constructs for HIF1A-HiBiT, diluted in carrier DNA. In parallel, HiBiT was tagged to the endogenous locus in HeLa cells using CRISPR/Cas9, and a clone was isolated. Untransfected cells, transfected cells and the endogenously tagged cell line were plated in 96-well plates and treated the following day for 4 hours with a titration of 1,10-phenanthroline. The Nano-Glo® HiBiT Lytic Reagent was added to all wells, and luminescence was measured after 10 minutes. Panel A. Background-subtracted luminescence shows the varying expression levels of HIF1A-HiBiT in transiently-transfected cells compared to expression from the endogenous promoter. Panel B. Data normalized to untreated cells shows how overexpression of HIF1A-HiBiT reduces the fold response from treatment with 1,10-phenanthroline. Error bars represent the standard deviation for n = 6.

//embed.widencdn.net/img/promega/whcbzd89vi/640px/14287MA-W.jpeg?u=7fvzhm

Conclusion

It is easy to focus on the sensitivity of your assay and forget that an assay’s results are only as sensitive as the detection level of the instrument. The combination of instrument and assay sensitivity provide the true level of detection for your experiment. Having an instrument with the sensitivity necessary to achieve the level of detection you need means you will need to use less sample, less enzyme and less catalyst to perform your experiments. You will also be able to detect fewer cells, lower levels of transcription and more subtle changes in molecular interactions. Sensitivity comparisons (LOD and LOQ) between commercially available detection instruments indicated GloMax® Discover and Explorer exhibited 1 to 2 logs better sensitivity of the instruments tested (Figure 1). The low instrument background and overall design means that the results detected for each well by the GloMax® instruments give you best chance to measure near physiological levels in luminescent assays.

How to Cite This Article

Promega Corporation Successful Luminescent Assays Need Sensitive Instruments. [Internet] September 2017; tpub_190. [cited: year, month, date]. Available from: http://www.promega.com/resources/pubhub/successful-luminescent-assays-need-sensitive-instruments/

Promega Corporation Successful Luminescent Assays Need Sensitive Instruments. Promega Corporation Web site. http://www.promega.com/resources/pubhub/successful-luminescent-assays-need-sensitive-instruments/ Updated September 2017; tpub_190. Accessed Month Day, Year.

GloMax and Nano-Glo are registered trademarks of Promega Corporation. Bio-Glo is a trademark of Promega Corporation.

Figures

Figure 1. Sensitivity of luminescent detection on GloMax® systems and other commercially available plate readers.

The Bio-Glo™ Luciferase Assay System was used to test instrument sensitivity, and the limit of detection (LOD) was calculated.

The Bio-Glo™ Luciferase Assay System was used to test instrument sensitivity, and the limit of detection (LOD) was calculated.

//embed.widencdn.net/img/promega/0xdvronuy9/640px/14496MA-W.jpeg?u=7fvzhm
Figure 2. Stabilizing HIF1A-HiBiT by incubation with the hypoxia mimetic 1,10-phenanthroline.

HeLa cells were transiently transfected with different amounts of CMV- or PGK-driven expression constructs for HIF1A-HiBiT, diluted in carrier DNA. In parallel, HiBiT was tagged to the endogenous locus in HeLa cells using CRISPR/Cas9, and a clone was isolated. Untransfected cells, transfected cells and the endogenously tagged cell line were plated in 96-well plates and treated the following day for 4 hours with a titration of 1,10-phenanthroline. The Nano-Glo® HiBiT Lytic Reagent was added to all wells, and luminescence was measured after 10 minutes. Panel A. Background-subtracted luminescence shows the varying expression levels of HIF1A-HiBiT in transiently-transfected cells compared to expression from the endogenous promoter. Panel B. Data normalized to untreated cells shows how overexpression of HIF1A-HiBiT reduces the fold response from treatment with 1,10-phenanthroline. Error bars represent the standard deviation for n = 6.

HeLa cells were transiently transfected with different amounts of CMV- or PGK-driven expression constructs for HIF1A-HiBiT, diluted in carrier DNA. In parallel, HiBiT was tagged to the endogenous locus in HeLa cells using CRISPR/Cas9, and a clone was isolated. Untransfected cells, transfected cells and the endogenously tagged cell line were plated in 96-well plates and treated the following day for 4 hours with a titration of 1,10-phenanthroline. The Nano-Glo® HiBiT Lytic Reagent was added to all wells, and luminescence was measured after 10 minutes. Panel A. Background-subtracted luminescence shows the varying expression levels of HIF1A-HiBiT in transiently-transfected cells compared to expression from the endogenous promoter. Panel B. Data normalized to untreated cells shows how overexpression of HIF1A-HiBiT reduces the fold response from treatment with 1,10-phenanthroline. Error bars represent the standard deviation for n = 6.

//embed.widencdn.net/img/promega/whcbzd89vi/640px/14287MA-W.jpeg?u=7fvzhm
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