Tags

Type your tag names separated by a space and hit enter

Lamp-based wavelength-resolved fluorescence detection for protein capillary electrophoresis: setup and detector performance.
Electrophoresis. 2010 Sep; 31(17):2861-8.E

Abstract

A lamp-based fluorescence detection (Flu) system for CE was extended with a wavelength-resolved (WR) detector to allow recording of full protein emission spectra. WRFlu was achieved using a fluorescence cell that employs optical fibres to lead excitation light from a Xe-Hg lamp to the capillary window and protein fluorescence emission to a spectrograph equipped with a CCD. A 280 nm band pass filter etc. together with a 300 nm short pass cut-off filter was used for excitation. A capillary cartridge was modified to hold the detection cell in a commercial CE instrument enabling WRFlu in routine CE. The performance of the WRFlu detection was evaluated and optimised using lysozyme as model protein. Based on reference spectral data, a signal-intensity adjustment was introduced to correct for transmission losses in the detector optics that occurred for lower protein emission wavelengths. CE-WRFlu of lysozyme was performed using BGEs of 50 mM sodium phosphate (pH 6.5 or 3.0) and a charged-polymer coated capillary. Using the 3-D data set, signal averaging over time and emission-wavelength intervals was carried out to improve the S/N of emission spectra and electropherograms. The detection limit for lysozyme was 21 nM, providing sufficient sensitivity to obtain spectral information on protein impurities.

Links

Publisher Full Text (DOI)

Authors+Show Affiliations

de Kort BJ
Department of Biomedical Analysis, Utrecht University, Utrecht, The Netherlands. B.J.deKort@uu.nl
de Jong GJ
No affiliation info available
Somsen GW
No affiliation info available

MeSH

AnimalsChickensElectrophoresis, CapillaryMuramidaseOptical FibersPhosphatesProteinsSensitivity and SpecificitySpectrometry, FluorescenceTemperature

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20715127

Citation

de Kort, Bregje J., et al. "Lamp-based Wavelength-resolved Fluorescence Detection for Protein Capillary Electrophoresis: Setup and Detector Performance." Electrophoresis, vol. 31, no. 17, 2010, pp. 2861-8.
de Kort BJ, de Jong GJ, Somsen GW. Lamp-based wavelength-resolved fluorescence detection for protein capillary electrophoresis: setup and detector performance. Electrophoresis. 2010;31(17):2861-8.
de Kort, B. J., de Jong, G. J., & Somsen, G. W. (2010). Lamp-based wavelength-resolved fluorescence detection for protein capillary electrophoresis: setup and detector performance. Electrophoresis, 31(17), 2861-8. https://doi.org/10.1002/elps.201000246
de Kort BJ, de Jong GJ, Somsen GW. Lamp-based Wavelength-resolved Fluorescence Detection for Protein Capillary Electrophoresis: Setup and Detector Performance. Electrophoresis. 2010;31(17):2861-8. PubMed PMID: 20715127.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Lamp-based wavelength-resolved fluorescence detection for protein capillary electrophoresis: setup and detector performance. AU - de Kort,Bregje J, AU - de Jong,Gerhardus J, AU - Somsen,Govert W, PY - 2010/8/18/entrez PY - 2010/8/18/pubmed PY - 2010/12/16/medline SP - 2861 EP - 8 JF - Electrophoresis JO - Electrophoresis VL - 31 IS - 17 N2 - A lamp-based fluorescence detection (Flu) system for CE was extended with a wavelength-resolved (WR) detector to allow recording of full protein emission spectra. WRFlu was achieved using a fluorescence cell that employs optical fibres to lead excitation light from a Xe-Hg lamp to the capillary window and protein fluorescence emission to a spectrograph equipped with a CCD. A 280 nm band pass filter etc. together with a 300 nm short pass cut-off filter was used for excitation. A capillary cartridge was modified to hold the detection cell in a commercial CE instrument enabling WRFlu in routine CE. The performance of the WRFlu detection was evaluated and optimised using lysozyme as model protein. Based on reference spectral data, a signal-intensity adjustment was introduced to correct for transmission losses in the detector optics that occurred for lower protein emission wavelengths. CE-WRFlu of lysozyme was performed using BGEs of 50 mM sodium phosphate (pH 6.5 or 3.0) and a charged-polymer coated capillary. Using the 3-D data set, signal averaging over time and emission-wavelength intervals was carried out to improve the S/N of emission spectra and electropherograms. The detection limit for lysozyme was 21 nM, providing sufficient sensitivity to obtain spectral information on protein impurities. SN - 1522-2683 UR - https://www.unboundmedicine.com/medline/citation/20715127/Lamp_based_wavelength_resolved_fluorescence_detection_for_protein_capillary_electrophoresis:_setup_and_detector_performance_ L2 - https://doi.org/10.1002/elps.201000246 DB - PRIME DP - Unbound Medicine ER -