Cover Image

Immobilized Metal Affinity Chromatography purification (IMAC) of his-tagged CTCF-ZN (zinc fingers domain) protein: a comparison between iminodiacetic acid (IDA) - based sepharose and nitriloacetic acid (NTA) - based agarose

Since its introduction, Immobilized Metal Affinity Chromatography (IMAC) has massive development by devising several variants of protocols and metal affinity-based techniques, seeking the best protein separation technique to produce high quality and quantity purified protein. This study utilized...

Full description

Saved in:
Bibliographic Details
Main Author: Samad, Maisarah Ab
Format: Monograph
Language:English
Published: Pusat Pengajian Sains Kesihatan, Universiti Sains Malaysia 2013
Subjects:
QD71-142 Analytical chemistry
Online Access:http://eprints.usm.my/58851/1/MAISARAH%20BINTI%20AB%20SAMAD%20-%20e.pdf
http://eprints.usm.my/58851/
Tags: Add Tag
No Tags, Be the first to tag this record!
id my.usm.eprints.58851
record_format eprints
spelling my.usm.eprints.58851 http://eprints.usm.my/58851/ Immobilized Metal Affinity Chromatography purification (IMAC) of his-tagged CTCF-ZN (zinc fingers domain) protein: a comparison between iminodiacetic acid (IDA) - based sepharose and nitriloacetic acid (NTA) - based agarose Samad, Maisarah Ab QD71-142 Analytical chemistry Since its introduction, Immobilized Metal Affinity Chromatography (IMAC) has massive development by devising several variants of protocols and metal affinity-based techniques, seeking the best protein separation technique to produce high quality and quantity purified protein. This study utilized two different types of metal affinity ligands with the aim of comparing the effectiveness of purification technique for Histagged CTCF-Zn (Zinc finger domain) protein. Iminodiacetic acid (IDA) and Nitriloacetic acid (NTA) were used to chelate the Ni2+ respectively forming the immobilized metal affinity ligand matrices. Different protocols were used in which column purification was applied in the IDA-based IMAC and batch purification for the latter. By modifying the imidazole concentration in the washing and elution buffers, same buffers were used for both IDA-based and NTA-based IMAC. Through the SDSPAGE and western blot analysis, the IDA-based IMAC demonstrates higher His-tag protein recovery and yield compared to NTA-based IMAC. Furthermore, the high protein retention featured by IDA caused low protein loss in the flow through and after washing. In contrast, NTA-based IMAC resulted in low protein affinity and more Histagged protein escaped before the elutions. The level of purity of the protein was relatively similar for both methods as the His-tagged protein purity was seen to be influenced by the structure of the protein and the imidazole concentration of the buffers. Pusat Pengajian Sains Kesihatan, Universiti Sains Malaysia 2013 Monograph NonPeerReviewed application/pdf en http://eprints.usm.my/58851/1/MAISARAH%20BINTI%20AB%20SAMAD%20-%20e.pdf Samad, Maisarah Ab (2013) Immobilized Metal Affinity Chromatography purification (IMAC) of his-tagged CTCF-ZN (zinc fingers domain) protein: a comparison between iminodiacetic acid (IDA) - based sepharose and nitriloacetic acid (NTA) - based agarose. Project Report. Pusat Pengajian Sains Kesihatan, Universiti Sains Malaysia. (Submitted)
institution Universiti Sains Malaysia
building Hamzah Sendut Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Sains Malaysia
content_source USM Institutional Repository
url_provider http://eprints.usm.my/
language English
topic QD71-142 Analytical chemistry
spellingShingle QD71-142 Analytical chemistry
Samad, Maisarah Ab
Immobilized Metal Affinity Chromatography purification (IMAC) of his-tagged CTCF-ZN (zinc fingers domain) protein: a comparison between iminodiacetic acid (IDA) - based sepharose and nitriloacetic acid (NTA) - based agarose
description Since its introduction, Immobilized Metal Affinity Chromatography (IMAC) has massive development by devising several variants of protocols and metal affinity-based techniques, seeking the best protein separation technique to produce high quality and quantity purified protein. This study utilized two different types of metal affinity ligands with the aim of comparing the effectiveness of purification technique for Histagged CTCF-Zn (Zinc finger domain) protein. Iminodiacetic acid (IDA) and Nitriloacetic acid (NTA) were used to chelate the Ni2+ respectively forming the immobilized metal affinity ligand matrices. Different protocols were used in which column purification was applied in the IDA-based IMAC and batch purification for the latter. By modifying the imidazole concentration in the washing and elution buffers, same buffers were used for both IDA-based and NTA-based IMAC. Through the SDSPAGE and western blot analysis, the IDA-based IMAC demonstrates higher His-tag protein recovery and yield compared to NTA-based IMAC. Furthermore, the high protein retention featured by IDA caused low protein loss in the flow through and after washing. In contrast, NTA-based IMAC resulted in low protein affinity and more Histagged protein escaped before the elutions. The level of purity of the protein was relatively similar for both methods as the His-tagged protein purity was seen to be influenced by the structure of the protein and the imidazole concentration of the buffers.
format Monograph
author Samad, Maisarah Ab
author_facet Samad, Maisarah Ab
author_sort Samad, Maisarah Ab
title Immobilized Metal Affinity Chromatography purification (IMAC) of his-tagged CTCF-ZN (zinc fingers domain) protein: a comparison between iminodiacetic acid (IDA) - based sepharose and nitriloacetic acid (NTA) - based agarose
title_short Immobilized Metal Affinity Chromatography purification (IMAC) of his-tagged CTCF-ZN (zinc fingers domain) protein: a comparison between iminodiacetic acid (IDA) - based sepharose and nitriloacetic acid (NTA) - based agarose
title_full Immobilized Metal Affinity Chromatography purification (IMAC) of his-tagged CTCF-ZN (zinc fingers domain) protein: a comparison between iminodiacetic acid (IDA) - based sepharose and nitriloacetic acid (NTA) - based agarose
title_fullStr Immobilized Metal Affinity Chromatography purification (IMAC) of his-tagged CTCF-ZN (zinc fingers domain) protein: a comparison between iminodiacetic acid (IDA) - based sepharose and nitriloacetic acid (NTA) - based agarose
title_full_unstemmed Immobilized Metal Affinity Chromatography purification (IMAC) of his-tagged CTCF-ZN (zinc fingers domain) protein: a comparison between iminodiacetic acid (IDA) - based sepharose and nitriloacetic acid (NTA) - based agarose
title_sort immobilized metal affinity chromatography purification (imac) of his-tagged ctcf-zn (zinc fingers domain) protein: a comparison between iminodiacetic acid (ida) - based sepharose and nitriloacetic acid (nta) - based agarose
publisher Pusat Pengajian Sains Kesihatan, Universiti Sains Malaysia
publishDate 2013
url http://eprints.usm.my/58851/1/MAISARAH%20BINTI%20AB%20SAMAD%20-%20e.pdf
http://eprints.usm.my/58851/
_version_ 1772812278537650176
score 13.211869

Similar Items