Introduction
In biochemical and molecular biology research, accurate quantitative analysis is the foundation of reliable experimental data. UV-Vis spectrophotometry has become the most fundamental and widely used analytical technique in biochemistry laboratories due to its non-destructive nature, simple operation, high sensitivity, and minimal sample preparation requirements.
From genomics to proteomics, from enzyme kinetics to cell biology, UV-Vis spectrophotometry permeates every field of biochemical research. STON Technologies' NanoQuant series spectrophotometers meet diverse biochemical analysis needs with their excellent optical performance.
Nucleic Acid Quantification
DNA and RNA Quantification:
Nucleic acids (DNA and RNA) have maximum absorption at 260nm. According to the Beer-Lambert law, nucleic acid concentration can be calculated by measuring A₂₆₀:
- 1 OD₂₆₀ = 50 μg/mL double-stranded DNA
- 1 OD₂₆₀ = 33 μg/mL single-stranded DNA
- 1 OD₂₆₀ = 40 μg/mL RNA
Purity Assessment:
The A₂₆₀/A₂₈₀ ratio is an important indicator of nucleic acid purity:
- Pure DNA: A₂₆₀/A₂₈₀ ≈ 1.8-2.0
- Pure RNA: A₂₆₀/A₂₈₀ ≈ 2.0-2.2
- Low ratio indicates protein or phenol contamination
NanoQuant Series Advantages: High wavelength accuracy (±0.5nm to ±0.1nm) ensures precise measurements at 260nm and 280nm. The NanoQuant1920's baseline flatness of ±0.0005A enables accurate detection of low-concentration nucleic acid samples.
Protein Quantification
UV-Vis spectrophotometry offers several well-established methods for protein quantification:
Direct UV Method (280nm):
Tyrosine and tryptophan residues in proteins exhibit characteristic absorption at 280nm. The A₂₈₀/A₂₆₀ ratio indicates nucleic acid contamination level.
BCA Method (562nm):
The bicinchoninic acid (BCA) method is one of the most popular protein quantification assays. Proteins reduce Cu²⁺ to Cu⁺ under alkaline conditions, and BCA forms a purple complex with Cu⁺ at 562nm. The NanoQuant series photometric accuracy of ±0.002A ensures excellent linearity across a wide concentration range.
Bradford Method (595nm):
Coomassie Brilliant Blue G-250's absorption maximum shifts from 465nm to 595nm upon protein binding, proportional to protein concentration. This method is quick, sensitive, and compatible with SDS and reducing agents.
Enzyme Kinetics
Enzyme kinetics studies are fundamental to understanding catalytic mechanisms. UV-Vis spectrophotometry enables real-time monitoring of substrate consumption or product formation rates:
- Catalase: Monitoring H₂O₂ consumption at 240nm
- Lactate Dehydrogenase (LDH): Monitoring NADH at 340nm
- Alkaline Phosphatase (ALP): Monitoring p-nitrophenol at 405nm
- β-Galactosidase: Monitoring ONPG hydrolysis at 420nm
The NanoQuant1920 double-beam system delivers photometric noise of only ±0.0003A (500nm@0A), accurately capturing initial enzyme reaction rates.
ELISA Assays
ELISA is a widely used immunoassay method in biomedical research. Chromogenic substrates (TMB, OPD, ABTS) produce colored products through enzymatic catalysis:
- TMB: measured at 450nm
- OPD: measured at 492nm
- ABTS: measured at 405nm
Cell Viability Assays (MTT/CCK-8)
The MTT and CCK-8 assays are classic methods for evaluating cell proliferation and cytotoxicity. MTT is reduced by mitochondrial enzymes in viable cells to purple formazan (measured at 570nm); CCK-8 is measured at 450nm. The NanoQuant series' wide linear photometric range (-0.301 to 6.0A) accurately covers detection from low to high cell densities.
Conclusion
UV-Vis spectrophotometry is a cornerstone technology in biochemical research. From nucleic acid and protein quantification to enzyme kinetics, from ELISA to cell viability assays, the NanoQuant series spectrophotometers provide precise quantitative analysis data with stable and reliable performance.
