The ultra trace nucleic acid quantitative analyzer (such as the ultra trace nucleic acid protein analyzer) mainly uses UV visible spectrophotometry to test proteins, estimating their concentration by measuring the absorbance of proteins at specific wavelengths (such as 280nm). The following are the specific methods and key steps for testing proteins:
1、 Testing principle
The tryptophan (Trp), tyrosine (Tyr) residues, and Cys Cys disulfide bonds in protein molecules exhibit significant absorption at a wavelength of 280nm. The instrument directly calculates the protein concentration (mg/ml) by measuring the absorbance (A280) of the protein solution at 280nm and combining it with Lambert Beer's law. This method does not require the construction of a standard curve, but it is necessary to ensure that the sample does not contain other impurities with strong absorption at 280nm, such as nucleic acids and phenolic substances.
2、 Operation steps
Instrument preparation
Connect the power supply, start the instrument, and preheat for 5-10 minutes to a stable state.
Select the "Protein A280" detection mode and set the optical path length (usually 10mm, but the instrument may automatically adjust).
Use high-purity distilled water or blank buffer for zero point calibration to eliminate background interference.
Sample processing
Sample size: It is recommended to use 2 μ L of sample (some instruments support 0.5-2 μ L micro detection) to ensure the formation of a stable liquid column.
Avoid bubbles: When sampling, gently touch the bottom of the detection hole and slowly release the liquid to prevent bubbles from affecting the absorbance measurement.
Surface tension: If the surface tension of the sample is low (such as containing detergent), the sample volume can be increased to 4 μ L to ensure the stability of the liquid column.
blank control
Take 2 μ L of blank solution (such as buffer) and add it to the lower base. Place the upper base down and click the "Blank" button to record the background absorbance.
Wipe the base to ensure there is no residual liquid.
sample testing
Take 2 μ L of the sample to be tested and add it to the lower base. Lower the upper base and click the "Sample" button.
The instrument automatically measures absorbance and calculates concentration, and the results are usually displayed in mg/ml.
High concentration samples: If the absorbance value exceeds the instrument range (such as light intensity<200), the sample needs to be diluted and retested.
Data recording and cleaning
Save the test results and export them in CSV or PDF format.
Wipe the base with a dust-free cloth to avoid cross contamination.
3、 Key precautions
Baseline calibration
The default baseline calibration wavelength is 340nm, which users can adjust as needed.
Calibration must be completed before sample testing, otherwise the spectral values will shift, resulting in concentration calculation errors.
Sample purity
Avoid samples containing nucleic acids, phenols, or high concentrations of salt ions, which may interfere with absorption at 280nm.
If impurity containing samples need to be detected, colorimetric methods (such as BCA, Bradford method) or dual wavelength (280nm/340nm) background subtraction can be used.
instrument maintenance
Light source: Avoid prolonged exposure and turn off the light source when not in use to extend its lifespan.
Monochromator: Regularly replace desiccants (such as silica gel) to prevent the dispersing element from getting damp and moldy.
Detector: Protect from direct sunlight or moisture and dust accumulation, and keep clean.
Environment: The instrument should be placed on a dry and stable workbench, away from air conditioning vents or direct sunlight, and the temperature should be controlled at 25 ℃± 2 ℃.
Special sample processing
Condensed sample: Slowly load with a 45 degree inclined surface against the wall to prevent overflow or interference from Newton's rings.
Polysaccharide substances: After detection, rinse the liquid circuit with ethanol reagent to avoid residue.
Oil sample: If there is an upward phenomenon, it needs to be separated by low-temperature centrifugation before testing.
4、 Application scenarios and advantages
Quick quantification: No complex preprocessing required, detection can be completed within 2-5 minutes, suitable for high-throughput screening.
Micro detection: Only 0.5-2 μ L of sample is required, saving precious samples such as clinical specimens and rare proteins.
Multi parameter analysis: Some instruments support simultaneous detection of A260 (nucleic acid), A280 (protein), and OD600 (bacterial concentration), achieving multi-component quantification.
Portability: Compact design, suitable for on-site testing or mobile laboratories.