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...
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| Main Author: | |
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| Format: | Monograph |
| Language: | English |
| Published: |
Pusat Pengajian Sains Kesihatan, Universiti Sains Malaysia
2013
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| Subjects: | |
| Online Access: | http://eprints.usm.my/58851/1/MAISARAH%20BINTI%20AB%20SAMAD%20-%20e.pdf http://eprints.usm.my/58851/ |
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| Summary: | 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. |
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