Effect of irradiation of ultraviolet on the quantitation method of enhanced green fluorescent protein

In last 30 years, green fluorescent protein (GFP) has changed from an unknown protein to a commonly used protein in bioscience application due to its visible fluorescence. As the usage of GFP increases, fluorescent detection and measurement devices are becoming more important. To detect and measure...

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Main Author:	Chee Heng, Hor
Format:	Undergraduates Project Papers
Language:	English English English
Published:	2015
Subjects:	QP Physiology
Online Access:	http://umpir.ump.edu.my/id/eprint/11019/ http://umpir.ump.edu.my/id/eprint/11019/ http://umpir.ump.edu.my/id/eprint/11019/1/FKKSA%20-%20HOR%20CHEE%20HENG%20%28CD8961%29.pdf http://umpir.ump.edu.my/id/eprint/11019/2/FKKSA%20-%20HOR%20CHEE%20HENG%20%28CD8961%29%20CHAP%201.pdf http://umpir.ump.edu.my/id/eprint/11019/3/FKKSA%20-%20HOR%20CHEE%20HENG%20%28CD8961%29%20CHAP%203.pdf

id	ump-11019
recordtype	eprints
spelling	ump-110192015-11-02T01:10:09Z http://umpir.ump.edu.my/id/eprint/11019/ Effect of irradiation of ultraviolet on the quantitation method of enhanced green fluorescent protein Chee Heng, Hor QP Physiology In last 30 years, green fluorescent protein (GFP) has changed from an unknown protein to a commonly used protein in bioscience application due to its visible fluorescence. As the usage of GFP increases, fluorescent detection and measurement devices are becoming more important. To detect and measure the GFP, a gel-based imaging system using a native polyacrylamide gel was developed. The ultimate aim of this study is to investigate the effect of ultraviolet (UV) light on the GFP quantitation method. In the research, enhanced GFP (EGFP) was expressed in Escherichia coli strain BL21(DE3) and purified using immobilized metal ions affinity chromatography. Different dilution of EGFP was prepared and their concentrations were determined by Lowry’s method using bovine serum albumin as the standard. The EGFP dilution samples were then loaded into a native polyacrylamide gel. After electrophoresis, fluorescent image of EGFP on the gel was captured using gel imaging system under different UV irradiation exposure period. The UV irradiation has a marked influence on the EGFP fluorescence intensity. The fluorescence intensity was increased as the UV exposure period increased from 5-35 s. However, the fluorescence intensity decreased when the exposure period was increased further. Highest fluorescence intensity happened at around 35 s of UV exposure. By using different concentration of purified EGFP, the photobleaching process followed a first order reaction with rates between 3712-8213 int/s. The linearity showed insignificant change and lied within 0.922-0.946. It became more reliable when the UV exposure time increases. However, UV exposure time affected the fluorescence intensity, it is better to choose around 35s as UV exposure time due to highest fluorescence intensity when using gel-based imaging method as quantitation method. Key words: GFP, EGFP, UV, gel-based imaging method, quantitation 2015-01 Undergraduates Project Papers NonPeerReviewed application/pdf en http://umpir.ump.edu.my/id/eprint/11019/1/FKKSA%20-%20HOR%20CHEE%20HENG%20%28CD8961%29.pdf application/pdf en http://umpir.ump.edu.my/id/eprint/11019/2/FKKSA%20-%20HOR%20CHEE%20HENG%20%28CD8961%29%20CHAP%201.pdf application/pdf en http://umpir.ump.edu.my/id/eprint/11019/3/FKKSA%20-%20HOR%20CHEE%20HENG%20%28CD8961%29%20CHAP%203.pdf Chee Heng, Hor (2015) Effect of irradiation of ultraviolet on the quantitation method of enhanced green fluorescent protein. Faculty of Chemical & Natural Resources Engineering, Universiti Malaysia Pahang. http://iportal.ump.edu.my/lib/item?id=chamo:91348&theme=UMP2
repository_type	Digital Repository
institution_category	Local University
institution	Universiti Malaysia Pahang
building	UMP Institutional Repository
collection	Online Access
language	English English English
topic	QP Physiology
spellingShingle	QP Physiology Chee Heng, Hor Effect of irradiation of ultraviolet on the quantitation method of enhanced green fluorescent protein
description	In last 30 years, green fluorescent protein (GFP) has changed from an unknown protein to a commonly used protein in bioscience application due to its visible fluorescence. As the usage of GFP increases, fluorescent detection and measurement devices are becoming more important. To detect and measure the GFP, a gel-based imaging system using a native polyacrylamide gel was developed. The ultimate aim of this study is to investigate the effect of ultraviolet (UV) light on the GFP quantitation method. In the research, enhanced GFP (EGFP) was expressed in Escherichia coli strain BL21(DE3) and purified using immobilized metal ions affinity chromatography. Different dilution of EGFP was prepared and their concentrations were determined by Lowry’s method using bovine serum albumin as the standard. The EGFP dilution samples were then loaded into a native polyacrylamide gel. After electrophoresis, fluorescent image of EGFP on the gel was captured using gel imaging system under different UV irradiation exposure period. The UV irradiation has a marked influence on the EGFP fluorescence intensity. The fluorescence intensity was increased as the UV exposure period increased from 5-35 s. However, the fluorescence intensity decreased when the exposure period was increased further. Highest fluorescence intensity happened at around 35 s of UV exposure. By using different concentration of purified EGFP, the photobleaching process followed a first order reaction with rates between 3712-8213 int/s. The linearity showed insignificant change and lied within 0.922-0.946. It became more reliable when the UV exposure time increases. However, UV exposure time affected the fluorescence intensity, it is better to choose around 35s as UV exposure time due to highest fluorescence intensity when using gel-based imaging method as quantitation method. Key words: GFP, EGFP, UV, gel-based imaging method, quantitation
format	Undergraduates Project Papers
author	Chee Heng, Hor
author_facet	Chee Heng, Hor
author_sort	Chee Heng, Hor
title	Effect of irradiation of ultraviolet on the quantitation method of enhanced green fluorescent protein
title_short	Effect of irradiation of ultraviolet on the quantitation method of enhanced green fluorescent protein
title_full	Effect of irradiation of ultraviolet on the quantitation method of enhanced green fluorescent protein
title_fullStr	Effect of irradiation of ultraviolet on the quantitation method of enhanced green fluorescent protein
title_full_unstemmed	Effect of irradiation of ultraviolet on the quantitation method of enhanced green fluorescent protein
title_sort	effect of irradiation of ultraviolet on the quantitation method of enhanced green fluorescent protein
publishDate	2015
url	http://umpir.ump.edu.my/id/eprint/11019/ http://umpir.ump.edu.my/id/eprint/11019/ http://umpir.ump.edu.my/id/eprint/11019/1/FKKSA%20-%20HOR%20CHEE%20HENG%20%28CD8961%29.pdf http://umpir.ump.edu.my/id/eprint/11019/2/FKKSA%20-%20HOR%20CHEE%20HENG%20%28CD8961%29%20CHAP%201.pdf http://umpir.ump.edu.my/id/eprint/11019/3/FKKSA%20-%20HOR%20CHEE%20HENG%20%28CD8961%29%20CHAP%203.pdf
first_indexed	2023-09-18T22:11:18Z
last_indexed	2023-09-18T22:11:18Z
_version_	1777415046034358272

Effect of irradiation of ultraviolet on the quantitation method of enhanced green fluorescent protein

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