SpectraMax ABS and ABS Plus Absorbance ELISA Microplate Readers

Absorbance

Learn all about absorbance detection – how it works, how it’s measured, and how it can be used to calculate concentration. We also provide information on common absorbance applications and assays including ELISAs, nucleic acid and protein quantitation, and microbial growth.

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Cell Health

Cell viability refers to the number of healthy cells in a population and can be evaluated using assays that measure enzyme activity, cell membrane integrity, ATP production, and other indicators. These methods can employ luminescent, fluorescent, or colorimetric readouts as indicators of general cell viability or even specific cellular pathways. Cytotoxicity and cell viability assays are often used to assess a drug or other treatment’s effect, and are valuable tools in the search for new therapeutics, as well as advancing our understanding of how normal cells function.

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Cell Viability, Cell Proliferation, Cytotoxicity Assays (MTT, XTT, MTS)

Colorimetric assays using tetrazolium salts like MTT, XTT, and MTS can be used to measure cell proliferation and cytotoxicity. For example, the MTT assay depends on the reduction of MTT by enzymes present in viable cells to form a blue formazan product that can be quantified by measuring the absorbance. The choice of assay may be based on the desired workflow and time required.

Read the application note to learn about some of these methods:

• Assess cell viability and proliferation with colorimetric readouts

DNA/RNA Quantitation

The absorbance of a DNA sample measured at 260 nm on a spectrophotometer or microplate reader can be used to calculate its concentration. Absorbance quantitation of DNA works on samples ranging from about 0.25 µg/mL to about 125 µg/mL in a microplate format.

Learn how absorbance is measured on our absorbance microplate readers with our featured app notes:

• DNA and RNA absorbance measurements using SpectraMax Microplate Readers
• Low-volume, high-throughput DNA and protein detection on the SpectraMax ABS Plus Microplate Reader

ELISA

Enzyme-linked immunosorbent assays (ELISAs) are used to measure the amount of a specific protein, using a microplate format, and results are most often detected via absorbance in the visible wavelength range. Chemiluminescent and fluorescent ELISA formats offer enhanced sensitivity for accurate quantitation of less abundant analytes.

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Endotoxin assays

Monitoring for contaminants is a critical step during the production process in the pharmaceutical and medical device industries. A frequent contaminant, endotoxin, can cause fever, inflammation, headache, nausea, and even death. Fortunately, endotoxins can be readily monitored using turbidimetric, colorimetric, or fluorometric assays.

These application notes describe the different types of endotoxin assays:

• Monitor for endotoxin with the SpectraMax ABS Plus Microplate Reader
• Microplate-based endotoxin testing in GLP/GMP environments using a colorimetric assay
• Detect endotoxin with the PyroGene Recombinant Factor C Assay

Enzyme kinetics, bacterial / microbial growth

Many biological experiments require monitoring bacterial growth or measuring enzymatic changes over long periods of time (hours, days or even weeks).

Here are a few featured application notes illustrating how to set up kinetic assays to measure bacterial growth using our instruments and software:

• Advanced kinetic analysis of a bacterial growth assay
• Multitask kinetic measurements of IPTG’s effects on protein expression and cell growth
• Measure long-term cell growth using a discontinuous kinetic reading

Micro-volume Applications

Quantitation of many sample types is accomplished through absorbance measurements in a cuvette or microplate. However, when material is scarce or samples are precious, quantitation of very low sample volumes is required. Instruments and accessories are available that enable quantitation in volumes as low as 2 µL, or even 0.5 µL.

Learn more about our micro-volume instruments and techniques:

• Direct protein quantitation using the SpectraDrop Micro-Volume Microplate
• Maximizing performance of the SpectraDrop Micro-Volume Microplate for DNA quantitation
• Nucleic acid quantitation and analysis using the QuickDrop Spectrophotometer

Microbiology and Contaminant

Microbes, including bacteria, have been estimated to make up about 15 percent of the earth’s biomass, and microbes in the human body outnumber human cells by 10 to 1. These microorganisms provide great benefit to us and are also vital to many fields of research from medicine to alternative energy production. On the other hand, monitoring for microbes and the toxic substances they produce is necessary to ensure the safety of pharmaceutical products. Scientists whose research relies on mammalian cells must carefully monitor these cultures for unwanted microbial contaminants to ensure that their experimental results are reliable.

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Nucleic Acid (DNA/RNA) Quantitation and Analysis

Nucleic acids are large biomolecules common to all known life forms. Deoxyribonucleic acid (DNA) consists of a double strand of pairs of nucleotides, while ribonucleic acid (RNA) is typically a single strand. In DNA, the nucleotides are adenine, cytosine, guanine, and thymine, while RNA contains uracil instead of thymine. DNA makes up the genetic material of all organisms, encoding the information cells need to synthesize proteins.

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Optical density measurements using PathCheck Technology

UV/VIS spectrophotometers and microplate readers differ fundamentally in their beam geometry. In spectrophotometers, samples are read through cuvettes or tubes with a horizontal, 1-cm light path, making assays based on extinction coefficients straightforward. In microplate readers, the vertical light beam results in a pathlength that depends on the volume of fluid in each well. This problem has been remedied with Molecular Devices PathCheck Technology, which automatically corrects optical density (OD) measurements to a 1-cm pathlength.

These application notes discuss the principles and uses of PathCheck Technology:

• Optical density measurements automatically corrected to a 1-cm pathlength with PathCheck Technology
• Phenolic compounds measurement in red wines using the SpectraMax Plus 384 Microplate Reader
• DNA and RNA absorbance measurements using SpectraMax Microplate Readers

Protein Detection, Quantitation and Analysis

Protein detection, quantitation, and analysis are central to the investigation of a wide variety of biological processes. Measuring the concentration of protein is necessary to processes ranging from protein purification and labeling to sample preparation for electrophoresis. Protein can be quantitated directly via absorbance at 280 nm, or indirectly using colorimetric (BCA, Bradford, etc.) or fluorometric methods offering advantages such as greater sensitivity. To identify and measure a specific protein within a complex sample, for example, serum or cell lysate, an ELISA may be used.

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Protein Quantitation (BCA, Bradford, Lowry assays)

Protein concentration can be measured directly, via absorbance at 280 nm in a UV spectrophotometer, or indirectly, using colorimetric methods such as BCA or Bradford assays.

Here are a few application notes on protein quantitation you may find of interest:

• Measure total protein in cell lysates with SpectraMax ABS Plus Microplate Reader
• Protein quantitation with the EMax Plus Microplate Reader
• Direct protein quantitation using the SpectraDrop Micro-Volume Microplate
• Streamline BCA-based protein quantitation on the SpectraMax iD5 reader