Hello everyone, the BCA instrument in the laboratory was broken recently, and I accidentally discovered that nanodrop can also measure protein concentration, which saves a lot of time! The principle of this method is : UV absorption
Friendly reminder: Due to the existence of the table, reading this tweet on a computer will provide better results.
| UV absorption method |
More sensitive 50~100mg |
Quick 5 to 10 minutes |
Light absorption of tyrosine and tryptophan residues in proteins at280nm |
various purines and pyrimidines; various nucleotides |
Used for detection of chromatography column effluent; nucleic acid absorption can be corrected |
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Select A280 wavelength to measure protein concentration
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Prepare the work area by ensuring that work surfaces are clean and that pipettes and test tubes used are cleaned.
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Use pure water or buffer instead of the protein sample to be measured for blank calibration. Place a small volume of pure water or buffer (the same volume as the sample to be measured) onto the NanoDrop measurement coverslip.
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Gently wipe the measuring cover slip with a sterile paper towel to remove surface impurities.
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Place the measurement cover slip into the measurement slot of the NanoDrop instrument and close the instrument lid.
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Select the measurement mode for protein concentration in the instrument software.
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Click the measurement button to start blank calibration and wait for a while until the calibration is completed.
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After calibration is complete, remove the blank sample and clean the measuring cover.
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Take a small amount of the protein sample to be measured (usually around 1-2 µl) and transfer it to a new NanoDrop measurement coverslip.
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Gently wipe the measuring cover slip with a sterile paper towel to remove surface impurities.
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Place the measurement cover slip into the measurement slot of the NanoDrop instrument and close the instrument lid.
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Click the Measure button to start measuring and wait for a while until the measurement is completed.
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Read the measurement results, including absorbance value and protein concentration. Typically, absorbance values are measured at a wavelength of 280 nanometers, which corresponds to the maximum absorption peak of the protein.
If you want to be more accurate, you can add your own standard product, measure the concentration of the standard product, and make a standard song.
Attached:
The five protein determination methods are compared as follows:
| method | Sensitivity | time | principle | interfering substances | illustrate |
| Kjedahl method | Low sensitivity, suitable for 0.2~1.0mg nitrogen, error is ±2% | Time consuming 8 to 10 hours | Convert protein nitrogen to ammonia, absorb with acid and titrate | Non-protein nitrogen (can be separated by precipitating proteins with trichloroacetic acid) | Used for accurate determination of standard protein content; less interference; too time-consuming |
| Biuret method (Biuret method) | Low sensitivity 1~20mg | Medium speed 20~30 minutes | Polypeptide bond + basic Cu2+® purple complex | Ammonium sulfate; Tris buffer; certain amino acids | is used for rapid determination, but is not very sensitive; different proteins develop similar colors; BCA chelates Cu+ as a chromogenic reagent to produce blue-violet color at 562nm< a i=2>There is an absorption peak |
| UV absorption method | More sensitive 50~100mg | Quick 5 to 10 minutes | Light absorption of tyrosine and tryptophan residues in proteins at280nm | various purines and pyrimidines; various nucleotides | Used for detection of chromatography column effluent; nucleic acid absorption can be corrected |
| Folin-phenol reagent method (Lowry method) | High sensitivity ~5mg | Slow speed 40~60 minutes | Biuret reaction; phosphomolybdic acid-phosphotungstic acid reagent is reduced by Tyr and Phe | Ammonium sulfate; Tris buffer; glycine; various thiols | It takes a long time; the operation must be strictly timed; the color depth changes with different proteins |
| Coomassie brilliant blue method (Bradford method) | The highest sensitivity is 1~5mg | Quick 5 to 15 minutes | When Coomassie Brilliant Blue dye binds to protein, its lmax changes from 465nm to 595nm. | Strong alkaline buffer; TritonX-100; SDS | The best method; less interfering substances; stable color; color depth changes with different proteins |