Immunodetection and purification of recombinant human heat shock protein 47

Heat shock protein 47 (HSP47) is a protein that resides in the endoplasmic reticulum, with interaction specific to its substrate namely collagen. Recombinant human HSP47 gene (hhsp47) was transformed into E. coli BL21(DE3) and grown in Luria-Bertani (LB) growth medium. Protein expression was carried...

Full description

Saved in:

Bibliographic Details
Main Author:	Ibrahim, Fatin Hazwani
Format:	Thesis
Published:	2014
Subjects:	QP Physiology
Tags:	Add Tag No Tags, Be the first to tag this record!

id	my-utm-ep.48474
record_format	uketd_dc
spelling	my-utm-ep.484742017-07-27T04:58:25Z Immunodetection and purification of recombinant human heat shock protein 47 2014 Ibrahim, Fatin Hazwani QP Physiology Heat shock protein 47 (HSP47) is a protein that resides in the endoplasmic reticulum, with interaction specific to its substrate namely collagen. Recombinant human HSP47 gene (hhsp47) was transformed into E. coli BL21(DE3) and grown in Luria-Bertani (LB) growth medium. Protein expression was carried out at 20oC with induction using 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). SDS-PAGE and Western blotting were performed to detect expression of hHSP47 in cell lysate and supernatant. The supernatant was then used for further applications such as immunoblotting and purification. In immunoblotting method optimisation, 5% (w/v) skimmed milk as blocking agent, 1 hour for blocking time, and 1:20000 secondary antibody concentration were empirically determined to have the most optimal detection with minimal background and high signal. For protein purification, affinity chromatography with the use of 1 mL HisTrapTM HP column was used. The purification yielded about 4 µM of around 80% purified hHSP47. SDS-PAGE analysis of the respective partially purified fraction shown a 47 kDa band, representing that hHSP47 was in monomeric form. Gelatin agarose pull down assay that aims to determine binding ability of hHSP7 to gelatin was performed before and after purification. The binding assay results indicates that hHSP47 still possesses its native folding after purification, leading to successful binding to gelatin 2014 Thesis http://eprints.utm.my/id/eprint/48474/ masters Universiti Teknologi Malaysia, Faculty of Bioscience and Medical Engineering Faculty of Bioscience and Medical Engineering
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
topic	QP Physiology
spellingShingle	QP Physiology Ibrahim, Fatin Hazwani Immunodetection and purification of recombinant human heat shock protein 47
description	Heat shock protein 47 (HSP47) is a protein that resides in the endoplasmic reticulum, with interaction specific to its substrate namely collagen. Recombinant human HSP47 gene (hhsp47) was transformed into E. coli BL21(DE3) and grown in Luria-Bertani (LB) growth medium. Protein expression was carried out at 20oC with induction using 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). SDS-PAGE and Western blotting were performed to detect expression of hHSP47 in cell lysate and supernatant. The supernatant was then used for further applications such as immunoblotting and purification. In immunoblotting method optimisation, 5% (w/v) skimmed milk as blocking agent, 1 hour for blocking time, and 1:20000 secondary antibody concentration were empirically determined to have the most optimal detection with minimal background and high signal. For protein purification, affinity chromatography with the use of 1 mL HisTrapTM HP column was used. The purification yielded about 4 µM of around 80% purified hHSP47. SDS-PAGE analysis of the respective partially purified fraction shown a 47 kDa band, representing that hHSP47 was in monomeric form. Gelatin agarose pull down assay that aims to determine binding ability of hHSP7 to gelatin was performed before and after purification. The binding assay results indicates that hHSP47 still possesses its native folding after purification, leading to successful binding to gelatin
format	Thesis
qualification_level	Master's degree
author	Ibrahim, Fatin Hazwani
author_facet	Ibrahim, Fatin Hazwani
author_sort	Ibrahim, Fatin Hazwani
title	Immunodetection and purification of recombinant human heat shock protein 47
title_short	Immunodetection and purification of recombinant human heat shock protein 47
title_full	Immunodetection and purification of recombinant human heat shock protein 47
title_fullStr	Immunodetection and purification of recombinant human heat shock protein 47
title_full_unstemmed	Immunodetection and purification of recombinant human heat shock protein 47
title_sort	immunodetection and purification of recombinant human heat shock protein 47
granting_institution	Universiti Teknologi Malaysia, Faculty of Bioscience and Medical Engineering
granting_department	Faculty of Bioscience and Medical Engineering
publishDate	2014
_version_	1747817399154900992

Immunodetection and purification of recombinant human heat shock protein 47

Similar Items