Hitrap protein g affinity column

To isolate murine IgG, serum samples prepared from immunized mice were diluted with an equal amount of PBS (pH 7.4) and passed through a sterile filter of 0.22 μm pore size (Millipore, Ireland). Preparations were subsequently passed through a HiTrap protein G Affinity column (GE Healthcare, USA) and IgG fractions were eluted with 0.1M glycine HCl buffer (pH 2.7). Protein contents were determined at a wavelength of 280 nm in a Biophotometer (Eppendorf, Germany) and sterile IgG fractions were adjusted to 1mg/ml before storage at -80°C.
In some experiments, IgG prepared from immunized mice was transferred into naive female Balb/c mice according to a protocol published previously [23 ], with modification. Briefly, sterile IgG was transferred into naive 8- to 10-week-old female Balb/c mice (400 μg IgG /20g bodyweight) by a single i.p. injection. Thereafter, mice were followed for seven days post antibody transfer.

ChIL-10 cDNA was sub-cloned into the vector, pKW06 (John Young, unpublished) to express the protein in mammalian cells with a C-terminal human IgG1 Fc tag. For screening purposes, to eliminate anti-human Ig antibodies, a V5His tagged chIL-10 was produced in the vector pKW08 (John Young and Tuanjun Hu, unpublished). ChIL-10 cDNAs (Rothwell et al., 2004 (link)) were expressed with the native signal peptide allowing secretion of the fusion proteins. Both constructs were expressed in COS-7 cells following transfection using the DEAE-dextran method (Rothwell et al., 2004 (link)). Recombinant chIL-10-Fc protein was purified using a HiTrap Protein G affinity column and the chIL-10-V5H6 protein with a HisTrap excel column (GE Healthcare Life Sciences).

Recombinant human DL4-Fc fusion protein was purchased from Sino Biological or manufactured in-house. The coding sequence of the extracellular domain of human DL4 was cloned upstream of the Fc portion of human IgG1 (including the hinge region) and inserted into a pIRESpuro2 mammalian expression plasmid (Clontech). HEK-293T cells were transfected using CaPO₄ transfection methods and stably integrated clones were selected using puromycin (2 µg/ml). Secreted DL4-Fc was purified from the supernatant using a HiTrap Protein G affinity column attached to the ATKAprime plus liquid chromatography system (both from GE Healthcare Life Sciences). Tissue culture 96-well plates were coated with DL4-Fc and murine VCAM-1-Fc (R&D Systems) overnight at 4 °C or for 2–4 h at room temperature. To coat, DL4-Fc and VCAM-1-Fc were diluted to 15 and 2.5 µg/ml, respectively, in 50 µl of phosphate-buffered saline (PBS), resulting in a coating concentration of ~24 ng/mm² of DL4-Fc and 4 ng/mm² of VCAM-1-Fc. For experiments with less DL4-Fc, the concentration was adjusted accordingly while maintaining a 50 µl volume of PBS. Wells were washed once with PBS prior to seeding cells to remove unbound protein.

The extracellular domains of chFLT3 or chFLT3s were sub‐cloned into pKW06 to produce a fusion protein with a C‐terminal human IgG1 Fc tag or pKW08 to produce a fusion protein with a V5HIS6 tag [36 (link)]. The constructs were named as pKW06/chFLT3, pKW06/chFLT3s, pKW08/chFLT3 and pKW08/chFLT3s. All constructs have their own signal peptides and were expressed in human embryonic kidney HEK293 cells as described elsewhere [37 (link)]. FLT3‐Fc was purified using a HiTrap Protein G affinity column (GE Healthcare Life Sciences). The purity and identity of FLT3‐Fc were confirmed by SDS‐PAGE and analysis of tryptic peptides by mass spectrometry (Dr Dominic Kurian, Proteomics and Metabolomics, The Roslin Institute) before immunization.

Monoclonal antibody purification and gold conjugation were performed as previously described [28 (link)]. Briefly, hybridoma culture supernatants were purified using a HiTrap Protein-G affinity column (GE, Fairfield, CT, USA) in an AKIA chromatography system.
The purified mAb was gold conjugated using the High Sensitivity Conjugation kit (80-nm Gold Nanospheres, nanoComposix, Inc., San Diego, CA, USA). Briefly, 70 µg 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 140 µg Sulfo-NHS were added to 1 mL Gold Nanospheres and incubated for 30 min at room temperature to activate carboxyl gold. The gold solution was washed twice with a 1 mL potassium phosphate reaction buffer (pH 7.4). The purified mAb (20 µg) was added to the gold solution and incubated for 1 h, followed by washing twice with a reaction buffer. The gold conjugated mAb was re-suspended in the conjugation buffer (PBS with 0.5% BSA, 0.5% casein, 1% tween 20, and 0.05% azide).

The coding sequence of the extracellular domain of human DLL4 was cloned upstream of a Histidine (His) tag followed by the Fc portion of human IgG3 (including the hinge region) along with a BirA recognition sequence (Avitag) at the C-terminus interspaced by Gly-Ser spacers (DL4-Fc, Fig. 1a). This construct was inserted into a pIRESpuro2 mammalian expression plasmid (Clontech, CA). The resulting plasmid was transfected into HEK-293T cells using a standard CaPO₄ transfection method and cells with a stably integrated plasmid were selected based on puromycin resistance (2 mg/mL). Cells were expanded in Freestyle 293 expression media (Thermo Fisher Scientific). Supernatant containing the HEK-293T cell-secreted DL4-Fc fusion protein was subjected to fraction purification using HiTrap Protein G affinity columns (GE Healthcare) attached to the ÄKTAprime plus (GE Healthcare) automated chromatography system. Eluted fractions that contained DL4-Fc protein, as determined by Western blot analysis, were pooled and protein concentration was determined by spectrophotometry.