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Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser-induced fluorescence detection.
Electrophoresis. 2009 Nov; 30(21):3780-5.E

Abstract

The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2-(diethylamino)ethanol and triethanolamine. A buffer solution containing 2-(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene-2,3-dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4-28) of proteins, compared with conventional absorbance detection.

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Publisher Full Text (DOI)

Authors+Show Affiliations

Kaneta T
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Fukuoka, Japan. kaneta@cstf.kyushu-u.ac.jp
Yamamoto D
No affiliation info available
Imasaka T
No affiliation info available

MeSH

AminesBoric AcidsElectrophoresis, CapillaryFluorescenceKineticsLasersNaphthalenesPolyethylene GlycolsProteinsSensitivity and Specificity

Pub Type(s)

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

Language

eng

PubMed ID

19862753

Citation

Kaneta, Takashi, et al. "Postcolumn Derivatization of Proteins in Capillary Sieving Electrophoresis/laser-induced Fluorescence Detection." Electrophoresis, vol. 30, no. 21, 2009, pp. 3780-5.
Kaneta T, Yamamoto D, Imasaka T. Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser-induced fluorescence detection. Electrophoresis. 2009;30(21):3780-5.
Kaneta, T., Yamamoto, D., & Imasaka, T. (2009). Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser-induced fluorescence detection. Electrophoresis, 30(21), 3780-5. https://doi.org/10.1002/elps.200900314
Kaneta T, Yamamoto D, Imasaka T. Postcolumn Derivatization of Proteins in Capillary Sieving Electrophoresis/laser-induced Fluorescence Detection. Electrophoresis. 2009;30(21):3780-5. PubMed PMID: 19862753.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser-induced fluorescence detection. AU - Kaneta,Takashi, AU - Yamamoto,Daisuke, AU - Imasaka,Totaro, PY - 2009/10/29/entrez PY - 2009/10/29/pubmed PY - 2010/1/5/medline SP - 3780 EP - 5 JF - Electrophoresis JO - Electrophoresis VL - 30 IS - 21 N2 - The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2-(diethylamino)ethanol and triethanolamine. A buffer solution containing 2-(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene-2,3-dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4-28) of proteins, compared with conventional absorbance detection. SN - 1522-2683 UR - https://www.unboundmedicine.com/medline/citation/19862753/Postcolumn_derivatization_of_proteins_in_capillary_sieving_electrophoresis/laser_induced_fluorescence_detection_ L2 - https://doi.org/10.1002/elps.200900314 DB - PRIME DP - Unbound Medicine ER -