UV/Vis spectroscopy for protein and DNA quantification
Low volume, high accuracy UV/Vis spectrometers
Proteins, DNA and RNA absorb light in the ultraviolet range in solution, which means you can apply UV/Vis spectroscopy to quantify their concentrations (Figure 1). Quantification with UV/Vis spectroscopy is called UV/Vis spectrometry and it uses Beer’s law, sometimes called Beer-Lambert law.
Figure 1. Protein dilution spectra and DNA dilution spectra.
How does UV/Vis Spectroscopy work?
The amount of light a protein, DNA or RNA sample absorbs is directly proportional to the concentration. Proteins absorb most at 280 nm while DNA and RNA peak at 260 nm. With Beer’s law you determine sample concentration from the absorbance at 280 nm or 260 nm (called A280 or A260), the known pathlength and the sample’s extinction coefficient or concentration factor. UV/Vis spectrophotometers perform these measurements and calculations automatically and without any additional reagents. The best spectrometers can measure at high-throughput and with low sample volumes.
Lunatic, Little Lunatic and Stunner spectrometers perform high-speed UV/Vis spectral analysis from 230–750 nm and 0.03–275 OD with just 2 μL of sample in fixed pathlength microcuvettes (Figure 2). The microcuvettes are part of unique microfluidic circuits that prevent any cross-contamination or evaporation while delivering highly accurate absorbance spectra. Having pathlengths that are fixed enables accuracy on Lunatic and Stunner to within 2% and precision within 1%.
High Lunatic and Stunner plates have 2 microcuvettes in each circuit, with pathlengths measuring 0.1 and 0.7 mm. The 0.1 mm microcuvette detects a high maximum absorbance while the larger microcuvette is more accurate for lower absorbances. These 2 different sized microcuvettes make the wide dynamic range possible. Lunatic or Stunner read 96 of these microfluidic circuits on their SBS-compatible plates in only 10 minutes. For more info on UV/Vis spectroscopy and Lunatic’s innovative technology, check out this tech note.
Looking for accuracy? Lunatic and Stunner quality has been proven with Reference Materials (RMs) from the National Institute for Standards and Technology (NIST). NIST has 3 RMs for testing UV/Vis spectrophotometers: a monoclonal antibody (NISTmAb), tryptophan and uracil – all at certified concentrations in defined buffers. Lunatic and Stunner measure these samples within 2% of the certified value (Figure 3), easily meeting both USP and Ph. Eur. guidelines.
UV/Vis spectroscopy is one of the oldest and most well-established methods of directly quantifying proteins, DNA and RNA. Unchained Labs pushes UV/Vis quantification to the next level with innovative optics, well-controlled consumables and honed analysis techniques.
Figure 2. High Lunatic plate circuit.
Figure 3. Lunatic and Stunner accuracy of NIST RMs.
All Unchained Labs UV/Vis Spectroscopy instruments deliver:
- Accurate and precise full-spectrum UV/Vis absorbance results from 230–750 nm
- Results from tiny 2 µL sample volumes
- Wicked fast reads
- Wide dynamic range from 0.03 to 275 OD
- IgG quantification from 0.02 – 200 mg/mL
- Nucleic acid quantification from 1.5 - 13750 ng/µL
- Microfluidic circuits to protect samples from evaporation and cross-contamination
- Fixed pathlength microcuvettes for maximum sensitivity and reproducibility
- Purpose-built applications for quantification of purified or non-purified samples
- Low maintenance and no clean-up
Check out our UV/Vis Spectroscopy instrument line-up below, with three solutions designed to solve protein and DNA quantification problems.
Lunatic makes batch quantification of biologics, DNA and RNA a no-brainer. All you need is 2 µL and 10 minutes to measure up to 96 samples. Run them straight-up, even at high concentrations, without ever diluting. Measurements are spot-on with accuracy within 2% and precision within 1% and are fully compliant with USP and Ph. Eur. guidelines. The SBS plate-based format means integrating with lab automation and liquid handlers is a snap. Lunatic gets UV/Vis quantification on the money every time. Just drop, load and read.
Stunner combines Lunatic’s plate-based, high-throughput UV/Vis accuracy and precision with dynamic light scattering for a one-of-a-kind combination. Pairing up these techniques checks concentration, hydrodynamic size, polydispersity and detection of aggregates off your to-do list in one shot. Sync up sample quantity and quality your way.
Little Lunatic is there for labs that have fewer samples and lower requirements. It uses a chip-based version of the Lunatic plate to get quick concentration measurements of up to 16 samples in 5 minutes. Researchers that just need a quick check of concentration can get what they need from this little guy and its glove-friendly touch screen.
Have a question or can’t wait to find out more?