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UV measurements in microplates suitable for high-throughput protein determination
Stefan Kreusch1, Sina Schwedler, Bärbel Tautkus
1Institute of Biochemistry I, Medical Faculty, Friedrich-Schiller University, Nonnenplan 2, D-07743 Jena, Germany.
Analytical Biochemistry
|February 28, 2003
Summary
This study presents a rapid UV spectrophotometric method for large-scale protein determination using microplates. The method offers high precision and accuracy without individual calibration, ideal for analyzing various protein samples.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Spectroscopy
Background:
- Accurate protein quantification is crucial in biological and biochemical research.
- Traditional protein assays can be time-consuming, require extensive sample preparation, and may involve sample loss.
- UV spectrophotometry offers a direct method for protein measurement, but challenges exist in large-scale applications and interference removal.
Purpose of the Study:
- To develop and validate a high-throughput UV spectrophotometric method for protein determination using microplates.
- To assess the precision, accuracy, and applicability of the method across a wide protein concentration range.
- To demonstrate the method's effectiveness in removing interfering substances and its utility in complex biological samples.
Main Methods:
- Utilized a SPECTRAmax PLUS reader with UVStar 96- and 384-well microplates for large-scale protein analysis.
- Employed a 96- or 384-parallel channel liquid handling technique and measured absorbance at 205, 215, and 280 nm.
- Incorporated a PathCheck option for determining absorbance coefficients and a parallel dialyzing device for interference removal.
Main Results:
- Achieved intra-assay precision better than 3% coefficient of variation (CV) for protein concentrations from 1 to 8000 µg/ml.
- Demonstrated accurate protein determination without individual calibration using an absorbance coefficient of 30 ml/(mg·cm) at 205 nm.
- Successfully analyzed samples (60-250 µl) without extensive treatment or sample loss, effectively removing interfering UV-absorbing substances like buffers and detergents.
Conclusions:
- The described UV spectrophotometric microplate method enables rapid, precise, and accurate large-scale protein determination.
- The method eliminates the need for individual protein calibration and efficiently removes interfering substances, simplifying sample analysis.
- This technique is suitable for diverse applications, including serum protein analysis, even in the presence of detergents like Triton X-100.