A new application on the NanoDrop One/One Microvolume UV-Vis spectrophotometer (Thermo Scientific) further enhances protein quantification. Using preprogrammed applications on the NanoDrop One, investigators can directly measure the amount of protein in a 2 µl sample. The addition of the A205 application offers deeper coverage and several advantages over the A280 application, including lower protein-to-protein variability and higher sensitivity. This, combined with patented sample-retention technology and low stray light performance, makes it even easier to accurately quantify proteins in a matter of seconds.1
Within the NanoDrop One Protein A205 application, users can choose between three different programs. First, the ?205=31 method is an extinction coefficient often used for peptides lacking tryptophan (Trp) and tyrosine (Tyr) residues. Second, the Scopes method is ideal for proteins containing a significant amount of Trp and Tyr residues and relies on an A280/A205 ratio. Finally, the Other = custom method ?205 1mg/mL is customizable for a wide range of proteins and peptides. This method is appropriate for pure preparations of proteins or peptides whose amino acid sequences are known, or peptides that lack aromatic amino acids.
To illustrate the effectiveness of the A205 application, Loughrey et al. (2016) made a sample of polymyxin: a cationic detergent antibiotic containing a peptide backbone but no Trp or Tyr residues. They measured the sample on both the NanoDrop One and the Evolution 300 UV-Vis spectrophotometers (Thermo Scientific). For measurements on the NanoDrop One instrument, they directly pipetted 2 μL of sample on the sample pedestal. They used a 10 mm quartz cuvette for measurements on the Evolution 300 spectrophotometer. The team found the NanoDrop One Protein A205 application produced consistent reads and is highly comparable with the Evolution 300, showing a standard deviation lower than .04.
Next, the team compared the Scopes and ?205=31 methods on the NanoDrop One. For this analysis, they chose three different proteins with varied amounts of aromatic residues: bovine serum albumin (BSA, 3 Trp and 21 Tyr residues), lysozyme (6 Trp and 3 Tyr residues) and polymyxin (no Trp, no Tyr).
The researchers found that the A205 quantification using the ?205=31 method gives comparable result when there are not many Trp or Tyr residues. They note that the number of Trp and Tyr residues has a large effect on the calculated concentration.
The authors also explain that the NanoDrop One is sensitive enough to also pick up on common protein buffers with absorbance at 205 nm. Investigators desiring to use the NanoDrop One should choose a protein buffer that does not contribute to the absorbance at 205 nm.
1. Loughrey, S., et al. (2016) “Using the NanoDrop One to quantify protein and peptide preparations at 205 nm,” Thermo Scientific application note 52774.