UV measurements in microplates suitable for high-throughput protein determination.
Abstract: An UV spectrophotometric method for protein determination using microplates is described. Using the SPECTRAmax PLUS reader, the UVStar 96- and 384-well microplates and a 96 or 384 parallel channel liquid handling technique, large-scale determinations can be performed with intraassay precision better than 3% CV (coefficient of variation) in the range from 1 to 8000 microg of protein/ml, measuring at 205, 215, and 280 nm and using different volume-dependent light-path lengths. Since the absorbance coefficient at 205 nm is found to be 30 ml/(mgxcm) for eight different proteins with a CV of 5.6% only with the Path Check option of the reader, protein concentration can be determined without any individual calibration. Samples in the volume range of 60-250 microl can be analyzed without time-consuming and expensive treatment and without sample loss. Using a special 96 or 384 parallel dialyzing device, low molecular weight substances which interfere with the analysis by their UV absorbance, such as buffers and detergents, can effectively be removed. Application examples for serum protein separation are also shown in the presence of the strongly UV absorbing detergent Triton X-100.
Publication Date: 2003-02-28 PubMed ID: 12605857DOI: 10.1016/s0003-2697(02)00460-8Google Scholar: Lookup
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- Journal Article
- Analytical Methods
- Biochemistry
- Biomechanics
- Biotechnology
- Cells
- Clinical Pathology
- Clinical Study
- Diagnosis
- Diagnostic Technique
- Disease Diagnosis
- Equine Health
- Immune Response
- Immune System
- In Vitro Research
- Laboratory Methods
- Physiology
- Protein
- Spectroscopy
- Veterinary Medicine
- Veterinary Procedure
- Veterinary Research
Summary
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This research article presents a method for large-scale protein determination using UV spectrophotometry in microplates, which is efficient in terms of time and cost, and offers a high degree of precision.
Overview of the Research and Method Described
- The research focuses on an UV spectrophotometric method for protein determination using microplates. This method allows for large-scale determinations and is noted for its high precision.
- Significant tools for the process include the SPECTRAmax PLUS reader, UVStar 96- and 384-well microplates, and a parallel channel liquid handling technique that can be adjusted for 96 or 384 channels.
Accuracy and Precision of the Method
- The researchers found that this method achieved an intraassay precision better than 3% CV (coefficient of variation) in the range from 1 to 8000 micrograms of protein per milliliter.
- A significant aspect of this method is that the protein concentration can be determined without any individual calibration.
Innovation in Procedure and Approach
- The process involves measuring at 205, 215, and 280 nanometers and utilizes different volume-dependent light-path lengths.
- The method allows for samples in the volume range of 60-250 microliters to be analyzed without any prerequisite costly and time-consuming treatment and without any sample loss.
Application of the Method
- A major highlight of this research is the use of a special 96 or 384 parallel dialyzing device. This device efficiently removes low molecular weight substances which interfere with the analysis by their UV absorbance, such as buffers and detergents.
- Application examples provided in the research include serum protein separation in the presence of the strongly UV absorbing detergent Triton X-100.
Cite This Article
APA
Kreusch S, Schwedler S, Tautkus B, Cumme GA, Horn A.
(2003).
UV measurements in microplates suitable for high-throughput protein determination.
Anal Biochem, 313(2), 208-215.
https://doi.org/10.1016/s0003-2697(02)00460-8 Publication
Researcher Affiliations
- Institute of Biochemistry I, Medical Faculty, Friedrich-Schiller University, Nonnenplan 2, D-07743 Jena, Germany.
MeSH Terms
- Animals
- Cattle
- Detergents / chemistry
- Horses
- Humans
- Microchemistry / methods
- Proteins / analysis
- Reproducibility of Results
- Sensitivity and Specificity
- Spectrophotometry, Ultraviolet / methods
Citations
This article has been cited 11 times.- Fukui T, Niikura T, Oda T, Kumabe Y, Nishiaki A, Kaigome R, Ohashi H, Sasaki M, Igarashi T, Oe K, Hamblin MR, Kuroda R. Safety of 222 nm UVC Irradiation to the Surgical Site in a Rabbit Model. Photochem Photobiol 2022 Nov;98(6):1365-1371.
- Welch D, Aquino de Muro M, Buonanno M, Brenner DJ. Wavelength-dependent DNA Photodamage in a 3-D human Skin Model over the Far-UVC and Germicidal UVC Wavelength Ranges from 215 to 255 nm. Photochem Photobiol 2022 Sep;98(5):1167-1171.
- Buonanno M, Welch D, Brenner DJ. Exposure of Human Skin Models to KrCl Excimer Lamps: The Impact of Optical Filtering(†). Photochem Photobiol 2021 May;97(3):517-523.
- Fukui T, Niikura T, Oda T, Kumabe Y, Ohashi H, Sasaki M, Igarashi T, Kunisada M, Yamano N, Oe K, Matsumoto T, Matsushita T, Hayashi S, Nishigori C, Kuroda R. Exploratory clinical trial on the safety and bactericidal effect of 222-nm ultraviolet C irradiation in healthy humans. PLoS One 2020;15(8):e0235948.
- Hanamura N, Ohashi H, Morimoto Y, Igarashi T, Tabata Y. Viability evaluation of layered cell sheets after ultraviolet light irradiation of 222 nm. Regen Ther 2020 Jun;14:344-351.
- Buonanno M, Randers-Pehrson G, Bigelow AW, Trivedi S, Lowy FD, Spotnitz HM, Hammer SM, Brenner DJ. 207-nm UV light - a promising tool for safe low-cost reduction of surgical site infections. I: in vitro studies. PLoS One 2013;8(10):e76968.
- Miyata Y, Chang L, Bainor A, McQuade TJ, Walczak CP, Zhang Y, Larsen MJ, Kirchhoff P, Gestwicki JE. High-throughput screen for Escherichia coli heat shock protein 70 (Hsp70/DnaK): ATPase assay in low volume by exploiting energy transfer. J Biomol Screen 2010 Dec;15(10):1211-9.
- Nishimura M, Shimizu Y, Fujii T, Okada Y, Yamamoto T, Ogawa Y, Koi T, Suehiro Y, Yamasaki T, Nishikawa J. Transient transfection using 222 nm far UV-C irradiation. Sci Rep 2025 May 14;15(1):16787.
- Buonanno M, Hashmi R, Petersen CE, Tang Z, Welch D, Shuryak I, Brenner DJ. Wavelength-dependent DNA damage induced by single wavelengths of UV-C radiation (215 to 255 nm) in a human cornea model. Sci Rep 2025 Jan 2;15(1):252.
- Buonanno M, Hashmi R, Petersen CE, Tang Z, Welch D, Shuryak I, Brenner DJ. Wavelength-Dependent DNA Damage Induced by Single Wavelengths of UVC Light (215 to 255 nm) in a Human Cornea Model. Res Sq 2024 Dec 3;.
- Nishikawa J, Tamura Y, Fujii T, Fukuda S, Yoneda S, Yamaura N, Takahashi S, Yamamoto T, Nojima J, Suehiro Y, Yamasaki T, Takami T. Far-Ultraviolet Light at 222 nm Affects Membrane Integrity in Monolayered DLD1 Colon Cancer Cells. Int J Mol Sci 2024 Jun 27;25(13).
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