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Preparation, Analysis and Use of an Affinity Adsorbent 127
2. Materials
2.1. Biomolecules
1. pM6: The GST-ZnF Cloning Vector, or pM6, was created by inserting a 319 base
pair (bp) segment, coding for the zinc finger gene, into pGEX-2TK (4969 bp,
accession number U13851.1) between the BamHI and EcoRI restriction sites 3 .
The pM6 plasmid employs antibiotic resistance as a selection marker. The pM6
plasmid encoding for the GST–ZnF was produced by Dr. David Palfrey at
the Department of Pharmaceutical Sciences, Aston University (UK) and kindly
supplied by Dr. Anna Hine.
2. GST–ZnF: The GST–ZnF molecule, comprised of the GST segment (27.7 kDa) and
fused to the zinc finger moiety (10.7 kDa), has a molecular weight of approximately
38.4 kDa.
3. Glutathione: Glutathione (=99%, MW 307) is a tripeptide made up of the amino
acids gamma-glutamic acid, cysteine and glycine. In this body of work, the reduced
form of glutathione is used as a covalently immobilized affinity ligand and in the
elution buffer (see Note 1).
2.2. Solid-Phase Adsorbent
1. Glutathione-Streamline: Streamline , acting as the solid-phase adsorbent, is
activated and the glutathione ligand immobilized to create the affinity adsorbent
as described in Subheading 3.
2.3. Buffers
Where required, adjust buffer pH using 1 M HCl or 1 M NaOH.
1. Phosphate-buffered saline (PBS): PBS is used as the equilibration and running
buffer. The buffer can be prepared by dissolving a PBS tablet in 200 ml of
deionized (DI) water to yield a buffer containing 10 mM phosphate buffer, 2.7 mM
potassium chloride and 137 mM sodium chloride, pH 7.4.
2. Elution buffer: 20 mM reduced glutathione, 100 mM Tris–HCl, pH 9.
3. Phosphate buffer (for GST enzyme activity assay): 1 M KH 2 PO 4 with 1 M K 2 HPO 4
added until pH 6.5 obtained.
4. GST enzyme activity assay reagent: 22 ml DI water, 2.5 ml phosphate buffer, 0.25
ml 100 mM reduced glutathione, 0.25 ml 100 mM 1-chloro-2,4-dinitrobenzene
(CDNB, ≥99%) (see Note 2).
5. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)
staining solution: 0.12% w/v Coomassie Blue, 48% v/v methanol, 60% v/v DI
water and 12% v/v glacial acetic acid.
6. SDS–PAGE de-staining solution: 70% v/v DI water, 20% v/v methanol and 10%
v/v glacial acetic acid.