Interference with Spectrophotometric Analysis of Nucleic Acids and Proteins by Leaching of Chemicals from Plastic Tubes

Abstract

Absorbance spectroscopy is routinely used to monitor the concentrations of nucleic acids and proteins within solutions and to assess changes in their structure caused by interaction with chemicals or other biomolecules. Biological samples used for such analyses are manipulated and stored in small microcentrifuge tubes (microtubes) composed of polypropylene plus several plastic additives. Here we demonstrate that normal handling of laboratory microtubes causes leaching of light-absorbing chemicals into biological samples that interfere with spectrophotometric measurements. The leached chromophores absorbed UV light strongly at 220 and 260 nm, which are the wavelengths normally used to detect and quantitate proteins and DNA. Some common laboratory techniques, including sonication and PCR, were particularly effective inducers of leaching. The magnitude of the increase in absorbance was dependent upon both exposure time and heat history, with greatest induction after tubes were warmed to temperatures at or above 37 degrees C. Mass spectrometry revealed that aqueous solutions stored in plastic microtubes accumulate a complex mixture of leached chemicals with molecular masses of 200-1400 Da. Leaching was ubiquitous among commercially available brands of microtubes, indicating a persistent source of error in biomolecule detection and concentration measurements.

Impact of size, secondary structure, and counterions on the binding of small ribonucleic acids to layered double hydroxide nanoparticles

“…Eppendorf Flex-Tubes (1.5 ml) that exhibit minimal leaching of polypropylene additives were purchased from Eppendorf. 31 All electrophoresis experiments were performed using 11 Â 14 cm Horizon gel rigs (Labrepco) and an Amersham/GE Healthcare EPS 601 power supply.…”

Factors affecting the association of single- and double-stranded RNAs with montmorillonite nanoclays

“…We have previously developed a centrifugation method for analyzing the binding of nucleic acids to montmorillonite in aqueous solutions using small polypropylene microcentrifuge tubes that exhibit minimal leaching of light-absorbing organic molecules upon warming [7,32]. Nucleic acids absorb UV light strongly at 260 nm and reduction of absorbance at this wavelength occurs when nucleic acids bound to clay particles are pelleted by centrifugation.…”

Characterization of a Cyclic Olefin Polymer Microcentrifuge Tube

“…Lewis et al (1) provided strong evidence that most brands of PP tubes contain leachable substances capable of absorbing light in the UV range. This could result in inaccuracies in the determination of the concentration and purity of nucleic acids or proteins in solution, especially when present at low concentrations.…”

“…A study by Lewis et al (1) provided clear evidence that leaching of these substances could potentially interfere with the determination of nucleic acid and protein concentration by UV spectrometry, particularly at a wavelength of 260 nm. The magnitude of leaching was greatly dependent on the nature of the solvent and also the time and temperature of incubation, but it appeared to be ubiquitous among the different brands of microcentrifuge tubes that were tested.…”