To quantitate GSTZ1 expression in the rat samples, a custom polyclonal antibody was raised against full-length expressed recombinant rat GSTZ1 (Cocalico Biologicals, Reamstown, PA). The antigen used to produce this antibody consisted of the rat GSTZ1 gene (NCBI accession#: NM_001109445.1), produced de novo with a N-terminal his-tag and subcloned into the pet21a vector (Bio Basic Inc., Markham, ON, CA). Protein was then produced in E. coli. and purified with a nickel column (ThermoFisher Scientific). Tissue samples, 300 μg of total cell-free homogenate protein per well, were separated on SDS-PAGE gels, transferred to nitrocellulose membranes and immunoblotted for GSTZ1 expression as described previously [2 (link), 17 (link)]. Due to variations in actin expression among tissues, equal loading was confirmed by Coomassie staining of the acrylamide gel following transfer to the nitrocellulose membrane. Inter-sample variability in total protein loading was less than 15% from the mean.
Nickel column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nickel column is a laboratory equipment used for the purification and separation of proteins, peptides, and other biomolecules. It utilizes the affinity between nickel ions and histidine-tagged proteins to facilitate the isolation and concentration of the target molecules.
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2 protocols using nickel column
1
Quantifying Rat GSTZ1 Protein Expression
2
Recombinant Expression and Purification of Sj-Cys
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DNA encoding the full-length Sj-Cys (GenBank accession# FJ617450) was synthesized by Zoobio Biotechnology, China, and then subcloned into yeast expression vector pPIC9k using EcoRI and NotI sites. The correct insert and reading frame of the constructed recombinant plasmid Sj-Cys/pPIC9k was confirmed by double-stranded DNA sequencing. The plasmid Sj-Cys/pPIC9k was linearized with SacI and then transformed into P. pastoris GS115 by electroporation. The expression of rSj-Cys with His-tag at C-terminus was induced with 0.5% methanol for 120 h. The expressed rSj-Cys secreted in the medium was purified with immobilized metal affinity chromatography (IMAC) using a nickel column (Thermo, USA) as previously described (Zhan et al., 2005 (link)). The concentration of purified rSj-Cys was measured using an enhanced BCA Protein Assay Kit (Beyotime, China). The purity of rSj-Cys was measured with SDS-PAGE and the His-tag protein was confirmed by Western blotting with the anti-His antibody.
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