Quantitation of Nucleic Acids and Proteins

@article{Gallagher2008QuantitationON,
  title={Quantitation of Nucleic Acids and Proteins},
  author={Sean R. Gallagher and Philippe R. Desjardins},
  journal={Current Protocols Essential Laboratory Techniques},
  year={2008},
  volume={5}
}
Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers in molecular biology. Two methods for direct absorbance measurements at 260 nm are described—the first is a traditional cuvette‐based method, and the second is a microvolume method that requires no cuvettes or capillaries. In addition, three fluorescence techniques using Hoechst 33258, ethidium bromide, and PicoGreen reagent are presented in this unit. These five procedures cover a… 

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References

SHOWING 1-10 OF 36 REFERENCES

Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy

TLDR
This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques, as well as three microvolume methods that use fiber optic technology in specialized cells or instrumentation to allow quantitation of DNA solutions.

Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution.

TLDR
A sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins, which is well suited for use with standard fluorescence microplate readers, fluorometers, and some laser scanners.

Spectrophotometric Determination of Protein Concentration

TLDR
This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution by comparison with a standard curve or published absorptivity values for that protein.

SPECTROPHOTOMETRIC AND TURBIDIMETRIC METHODS FOR MEASURING PROTEINS

Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity.

TLDR
RNA A260/280 ratios are found to be more reliable and reproducible when these spectrophotometric measurements were performed at pH 8.0-8.5 and the ability to detect protein contamination was significantly improved when RNA wasSpectrophotometrically analyzed in an alkaline solution.

Colorimetric protein assay techniques

TLDR
It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample.