Determination of isoelectric focusing method for proteomic analysis of purple variety of orthosiphon stamineus

An ancient medicinal plant known as Orthosiphon stamineus purple variety exhibiting multiple remedial effects by the bioactive compound had been reported. However, no study on its protein separation work was documented. Indeed, two-dimensional (2D) electrophoresis systems have few major issues such...

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Main Author:	Zulkifli, Zetty Amirah
Format:	Thesis
Language:	English
Published:	2017
Subjects:	TP Chemical technology
Online Access:	http://eprints.utm.my/id/eprint/79093/1/ZettyAmirahZulkifliMFBME2017.pdf
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spelling	my-utm-ep.790932018-09-27T06:07:36Z Determination of isoelectric focusing method for proteomic analysis of purple variety of orthosiphon stamineus 2017-07 Zulkifli, Zetty Amirah TP Chemical technology An ancient medicinal plant known as Orthosiphon stamineus purple variety exhibiting multiple remedial effects by the bioactive compound had been reported. However, no study on its protein separation work was documented. Indeed, two-dimensional (2D) electrophoresis systems have few major issues such as complex time-consuming procedure and poor reproducibility result. Therefore, this proteomic research can be utilized as preliminary platform in identification of protein responsible for the production of bioactive compound and its application as remedies. Different sample require different voltage of isoelectric focusing (IEF) to separate the protein due to its isoelectric point (pI) hence this study was conducted in order to determine the best IEF method producing reproducible protein spots and to determine the protein spot distribution and pattern from 2D proteomics of O. stamineus. Initially, protein extraction utilizing unmodified and modified (1 mL of 80% methanol in 0.1M ammonium acetate, 0.6 mL of each phenol and SDS buffer in 5 % ß-mercaptoethanol, and 0.15mL lysis buffer) phenol extraction with three preliminary washes were performed. The extracted protein was then quantified by Bradford Assay whereas its quality was checked via Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE). The modified extraction method resulted in higher spots distribution compared to the unmodified method. In this study, three different IEF method of method 1 (2000V), method 2 (4000V), and method 3 (8000V) were applied before the protein spots pattern and distribution were analysed further. It was found that method 2 was the best IEF method for this plant as 2-fold higher reproducible protein spots were produced. In fact, 15 proteins were expected to be associated with the protein spots obtained that may have high relation with photosynthesis and antioxidant activity. The findings suggested that numerous valuable proteins are expressed in O. stamineus. Hence, the proteins mechanism and properties should be revealed to serve the medical sector in the future. 2017-07 Thesis http://eprints.utm.my/id/eprint/79093/ http://eprints.utm.my/id/eprint/79093/1/ZettyAmirahZulkifliMFBME2017.pdf application/pdf en public http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:108142 masters Universiti Teknologi Malaysia, Faculty of Biosciences and Medical Engineering Faculty of Biosciences and Medical Engineering
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
language	English
topic	TP Chemical technology
spellingShingle	TP Chemical technology Zulkifli, Zetty Amirah Determination of isoelectric focusing method for proteomic analysis of purple variety of orthosiphon stamineus
description	An ancient medicinal plant known as Orthosiphon stamineus purple variety exhibiting multiple remedial effects by the bioactive compound had been reported. However, no study on its protein separation work was documented. Indeed, two-dimensional (2D) electrophoresis systems have few major issues such as complex time-consuming procedure and poor reproducibility result. Therefore, this proteomic research can be utilized as preliminary platform in identification of protein responsible for the production of bioactive compound and its application as remedies. Different sample require different voltage of isoelectric focusing (IEF) to separate the protein due to its isoelectric point (pI) hence this study was conducted in order to determine the best IEF method producing reproducible protein spots and to determine the protein spot distribution and pattern from 2D proteomics of O. stamineus. Initially, protein extraction utilizing unmodified and modified (1 mL of 80% methanol in 0.1M ammonium acetate, 0.6 mL of each phenol and SDS buffer in 5 % ß-mercaptoethanol, and 0.15mL lysis buffer) phenol extraction with three preliminary washes were performed. The extracted protein was then quantified by Bradford Assay whereas its quality was checked via Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE). The modified extraction method resulted in higher spots distribution compared to the unmodified method. In this study, three different IEF method of method 1 (2000V), method 2 (4000V), and method 3 (8000V) were applied before the protein spots pattern and distribution were analysed further. It was found that method 2 was the best IEF method for this plant as 2-fold higher reproducible protein spots were produced. In fact, 15 proteins were expected to be associated with the protein spots obtained that may have high relation with photosynthesis and antioxidant activity. The findings suggested that numerous valuable proteins are expressed in O. stamineus. Hence, the proteins mechanism and properties should be revealed to serve the medical sector in the future.
format	Thesis
qualification_level	Master's degree
author	Zulkifli, Zetty Amirah
author_facet	Zulkifli, Zetty Amirah
author_sort	Zulkifli, Zetty Amirah
title	Determination of isoelectric focusing method for proteomic analysis of purple variety of orthosiphon stamineus
title_short	Determination of isoelectric focusing method for proteomic analysis of purple variety of orthosiphon stamineus
title_full	Determination of isoelectric focusing method for proteomic analysis of purple variety of orthosiphon stamineus
title_fullStr	Determination of isoelectric focusing method for proteomic analysis of purple variety of orthosiphon stamineus
title_full_unstemmed	Determination of isoelectric focusing method for proteomic analysis of purple variety of orthosiphon stamineus
title_sort	determination of isoelectric focusing method for proteomic analysis of purple variety of orthosiphon stamineus
granting_institution	Universiti Teknologi Malaysia, Faculty of Biosciences and Medical Engineering
granting_department	Faculty of Biosciences and Medical Engineering
publishDate	2017
url	http://eprints.utm.my/id/eprint/79093/1/ZettyAmirahZulkifliMFBME2017.pdf
_version_	1747818145072021504

Determination of isoelectric focusing method for proteomic analysis of purple variety of orthosiphon stamineus

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