Monday, February 22, 2010

Micro-Volume Spectrophotometric Analysis

Micro-volume nucleic acid quantification using short pathlength spectrophotometric devices, such as NanoDrop, simply workflows through omission of dilution steps and conserve samples by using only 1-2 µL of sample, which may even be retained after analysis. The molar absorptivity of dsDNA and RNA at 260 nm is consistent whatever the amount or proportions of purine or pyrimidine bases and sufficient for accurate quantification of samples ranging from a few decades of ng/µL to thousands of ng/µL. This effectively covers the expected dsDNA or RNA concentration isolated from genomic DNA or RNA kits from a wide range of samples and also the isolation of plasmids on the concentration end.

Protein micro-volume spectrophotometric analysis is more problematic. The molar absorptivity of proteins at 280 nm is dependent on the relative amounts and proportions of amino acids with aromatic side chains, such as phenylalanine, tyrosine and tryptophan. The absorptivity of these side chains can also be affected by the 3° structure of the protein. Furthermore, sensitivity at 280 nm is typically limited to protein concentrations ranging from a few tenths of mg/mL to a few decades of mg/mL – a concentration range that is an order of magnitude higher that for nucleic acids. Fortunately, for most samples (i.e. cells, tissues), total protein is about an order of magnitude higher than total RNA.

Do you perform spectrophotometric micro-volume analysis? If so, which device do you use: NanoDrop, NanoVue, Epoch/Take3 or another? Do you use the device for nucleic acid or protein determinations, or both?

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