Protein and Nucleic Acid Detection

Protein and Nucleic Acid Detection

Quantitation of nucleic acids and protein in a microplate format offers higher throughput and automated calculation of results compared to other methods.

An accurate assessment of nucleic acid quantity and purity is essential for many assays in molecular biology and genetics. Quantitative PCR, molecular cloning, forensics, and next-generation sequencing are some of the techniques that are strongly affected by the amount and quality of starting DNA or RNA template. In some situations, such as working with degraded samples or in forensics, it is important to get a reliable measurement from a very small amount of sample. An ideal DNA/RNA quantitation instrument would be sensitive enough to work with very low sample amounts, as well as scalable for high-throughput assays. Two methods of quantifying nucleic acids are widely used: spectrophotometric reading of optical density, which is typically done on single samples but can also be performed in microplates, and fluorescent dyes that react with nucleotides, which are best for high-throughput assays in a microplate format.

  • 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 works on samples ranging from about 0.25 ug/mL to about 125 ug/mL in a microplate format. Some instrumentation enables the quantitation of very small sample volumes, as little as 2 uL. When greater sensitivity is required, fluorescence methods allow quantitation of as little as a few picograms of DNA.

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    Mycoplasma Monitoring

    Mycoplasma, the smallest and simplest of the prokaryotes, are common contaminants of cell cultures. Symptoms of mycoplasma contamination include a reduction in the rate of proliferation and changes in cellular responses, including gene expression. Mycoplasma cannot be detected by simply examining cell cultures under a microscope, so a variety of methods such as fluorescence- and luminescence-based assays have been developed to enable researchers to monitor for contamination.

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  • Kinase/Phosphatase Assays

    Kinases are one of the most critical targets in drug discovery today. These enzymes are key components in cellular signaling pathways, and disruption to their function causes a variety of diseases, such as metabolic diseases, certain cancers, and cardiac disease. The functionally related phosphatases and phosphodiesterases are also important screening targets.

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    Protein Quantitation (Bradford, Lowry, BCA, DC)

    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. Absorbance quantitation is easy to do as it does not require additional reagents, but colorimetric methods offer greater sensitivity and are often preferred when samples are precious. Both can be performed in a UV-vis spectrophotometer or in an absorbance microplate reader.

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  • Enzyme Activity

    Enzyme activity can be monitored in real time using a chromogenic substrate which, upon its addition to the enzyme, produces a change in color that is detectable on an absorbance microplate reader. Using an instrument with onboard liquid handling capability, substrate can be added automatically while the reaction is monitored, enabling detection of the earliest part of the reaction and thus more accurate estimates of binding affinity.

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    Western Blot Detection

    Western blotting is among the most common methods employed for the detection and quantitation of specific proteins. In this method, protein sample, e.g. from a cell lysate, is first separated by size on an SDS-PAGE gel. Proteins are then transferred to a nitrocellulose or PVDF membrane, which is probed with antibody specific to the protein of interest. Various techniques are used to detect proteins on western blot membranes including fluorescence and chemiluminescence.

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  • Fluorescent Protein Detection

    Fluorescent proteins have become enormously popular as tools for monitoring biological events in vivo. In addition to green fluorescent protein (GFP) from the jellyfish Aequorea victoria, there are now numerous others available from other species of jellyfish and reef coral. These proteins can be expressed in a diverse range of cells and organisms, where they are used to track many cellular processes, including protein synthesis and translocation, gene induction, and cell lineage.

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    SNP Genotyping

    Genotyping is a process for analyzing genetic differences among individuals by examining their DNA sequences. Single nucleotide polymorphisms (SNPs) are one of the most common types of genetic variation, consisting of a single nucleotide mutation at a specific locus. SNP genotyping has proven very useful in identifying disease-related mutations in various species, and as a result many techniques for SNP detection have been developed.

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    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. Chemiluminescent and fluorescent ELISA formats offer enhanced sensitivity for accurate quantitation of less abundant analytes.

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