Absorbance

How does absorbance detection work?

Spectrophotometers and absorbance plate readers measure how much light is absorbed by a sample. Microplate readers that are capable of detecting light in the ultraviolet (UV) range can be used to determine the concentration of nucleic acids (DNA and RNA) or protein directly, without the need for sample labeling. Light of a certain wavelength, dependent on the material being measured, is passed through a sample, and a detector on the other side of the microplate well measures how much of the original light was absorbed by the sample in the well.

What is absorbance measured in?

Absorbance is measured using a spectrophotometer or microplate reader, which is an instrument that shines light of a specified wavelength through a sample and measures the amount of light that the sample absorbs.

Spectrophotometer vs. Plate Reader

A standard spectrophotometer measures absorbance one sample at a time, typically placed in a cuvette through which light is sent horizontally. An absorbance plate reader offers higher throughput and can measure the absorbance of samples in microplates, usually 96-well or even 384-well, by sending light through each well vertically.

Learn more about Absorbance plate readers  

How to run an absorbance ELISA protocol?

An ELISA, or enzyme-linked immunosorbent assay, is a method used to quantitatively detect an antigen within a sample. Most ELISAs are run in microplates, with the bottom of the microplate wells serving as the solid surface to which an antigen of interest attaches, either directly or via an antibody. An enzyme conjugate, which reacts with substrate to produce a colored solution, is used to detect the antigen. A microplate washer is used to wash away non-specific, unbound material in the wells, and an absorbance ELISA plate reader detects the color change produced when target antigen is present.

Learn more about ELISAs  

How does stray light affect my optical density (OD) reading?

Stray light is a general term for unwanted light reaching the detector of an instrument. The effect of stray light on an absorbance reading is often an unexpectedly low OD; the absorbance measured is lower than the true absorbance of the sample. The impact is usually on linearity and is greatest when measuring ODs above 2.0-2.5. Stray light is not user-correctable and is usually not caused by user error, with possible causes including optical components like degraded excitation filters. Spectrophotometers and absorbance plate readers typically incorporate features to minimize stray light.

• Optical density measurements automatically corrected to a 1-cm pathlength with PathCheck Technology

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

ELISA / Immunoassays (Quantifying antigens, antibodies, cytokines...)

Enzyme-linked immunosorbent assays (ELISAs) are used to measure the amount of a specific protein, typically in a microplate format, and results are most often detected via absorbance in the visible wavelength range.

Here are several application notes about ELISAs you may find of interest:

• Quantitate interleukin-8 concentrations on the SpectraMax ABS Plus Microplate Reader with the SimpleStep ELISA kit
• High-throughput melamine detection with Romer Labs AgraQuant Melamine Assays and Molecular Devices Absorbance readers
• Quantifying gluten in beer using an ASBC-approved ELISA method

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

Customer story: Absorbance plate reader tests DNA/RNA-synthesizers

OligoMaker ApS uses the SpectraMax 190 reader to test DNA/RNA-synthesizers

"We use the SpectraMax 190 as an important step in testing the DNA/RNA-synthesizers we build. We have to quantify the primers and probes to determine how much DNA/RNA is synthesized on the DNA/RNA Synthesizer.”

Read more  

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

Customer story: Absorbance plate readers used in biofilm research

Université Catholique de Louvain uses our SpectraMax absorbance and multi-mode readers to help fight biofilms

“Another great advantage” says Wafi Siala of the UCL “is the large choice of microplate sizes from 6 to 384 wells which the SpectraMax M series offers.”

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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

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 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