Protein g hp spin trap column

mAb NZ-1, described above, was affinity purified using a Protein G HP spin trap column (GE Healthcare, Cat.#28903134) and concentrated using an ultra-centrifugal filter (EMD Millipore, Cat.# UFC905024) [27 (link)]. The concentration was measured by a Nanodrop 1000 spectrophotometer (ThermoScientific) and 0.67 μg added to 4x10⁴ F-NHDFs or CC-NHDFs in 200 μl of medium. The preparations were incubated at room temperature for 20 minutes. The contents of each tube were then divided equally between two collagen-coated wells. As controls, mAb in 200 μl PBS was processed similarly in the absence of cells. The plates were then incubated for 30 minutes at 5% CO₂ at 37°C. An MDA-MB-231/Matrigel mixture was then cast over the fibroblast cell layer as described above. Images were acquired daily for four days using the 10X objective of a Zeiss Axiovert 100 microscope.

The purification of serum total IgG (tIgG) was performed on the Protein G HP Spin Trap column as described by the manufacturer (GE Healthcare, USA). The tIgG samples were eluted at pH 2.5, immediately neutralized, dialyzed against phosphate buffered solution (PBS, 0.1% NaN₃), and stored at +4°C until being tested. About 8.5 mg of IgG was obtained from 1 mL of serum applied onto the Protein G Sepharose column. To obtain the IgG-depleted serum we used the same method on the Protein G HP Spin Trap column, except the serum volume applied to the Protein G column was three times lower, and the complete depletion of IgG was controlled using the Easy-Titer IgG Assay Kit (Thermo Scientific, USA).

The antisera against the recombinant Leishmania prohibitins were obtained in mice after intramuscular injection with 50 μg of Leish r PHB1 and in rabbits with 500 μg of Leish r PHB2 with complete Freund’s adjuvant. This first injection was followed by three more injections applied every 10 days with the same antigens plus incomplete Freund’s adjuvant. The animals were exsanguinated two weeks after the final injection. The sera were checked by ELISA in multi-well plates coated with 10 μg/well of each recombinant PHB in 0.1 M carbonate/bicarbonate coating buffer (pH 8.6). The plates were blocked with 2% skimmed milk (Molico, Nestlé) in PBS (pH 7.2) containing 0.05% Tween 20 (Merck KGaA, Darmstadt, Germany). Sera with titres above 1:800 were pooled and stored at -80 °C until use.
The IgGs from the two immunosera were purified by affinity chromatography using a Protein G HP Spin Trap column (GE Healthcare), and then the specific antibodies were obtained by immunoabsorption on PVDF strips containing the purified recombinant proteins. The specific IgG antibodies were recovered from the strip with 0.1 M glycine buffer at pH 4.5; the pH was restored immediately to neutrality by adding 1 M Tris-HCl buffer, pH 8.0, supplemented with 0.1% purified bovine albumin and stored at -80 °C.