nickel affinity column  (GE Healthcare)

Journal: Animal reproduction science

Article Title: Recombinant peptide reverses cryo-capacitation in ram sperm and improves in vitro fertilization

doi: 10.1016/j.anireprosci.2019.05.016

Figure Lengend Snippet: Cloning, gene expression and purification of TrxA-FNIIx4-HiS6; A. cDNA encoding four tandem FNII repeats from Epididymal Sperm Binding Protein 1 precursor (ELSPBP1) was amplified from a bovine epididymal cDNA library and cloned into the expression vector pET-32 Ek/LIC (Novagen, 5.917 bp); Construction included a thioredoxin tag (TrxA) at the N-terminal and a 6 Histidine tag (Hisx6) at the C-terminal; B. E. coli Rosetta cells were transformed with the construction FNIIx4-pET-32 Ek; Electrophoretic profile using SDS–PAGE indicates the soluble (S) and insoluble (I) fractions of E. coli cells after 16 h induction (+IPTG T16) with 0.1 mM IPTG at 18 °C; A soluble fraction of cells without induction is depicted (control); TrxA-FNIIx4-HiS6 protein solubilized from inclusion bodies was refolded and purified using a HiTrap nickel affinity column (Purification); Molecular weight marker is depicted at left; Arrow indicates the expected size for the protein (38 kDa)

Article Snippet: The TrxA-FNIIx4-HiS 6 purification was conducted using a HiTrap nickel affinity column (GE Healthcare) equilibrated with binding buffer.

Techniques: Clone Assay, Expressing, Purification, Binding Assay, Amplification, cDNA Library Assay, Plasmid Preparation, Transformation Assay, SDS Page, Affinity Column, Molecular Weight, Marker