br Size exclusion chromatography SEC separations were done
Size exclusion chromatography (SEC) separations were done using
For the immune affinity subtraction procedure, the coupling/ washing/binding buffer consisted in a phosphate buffered saline (PBS) solution (137 mM NaCl, 2.7 mM KCl, 10 mM phospate, pH 7.4)
purchased from Fisher Scientific. The blocking buffer solution (etha-nolamine 50% in binding buffer) was prepared with ethanolamine, 99%, from Acros Organics (Geel, Belgium) and the elution buffer (60 mM citric KIN59 pH 2.2–3.0) with citric acid, ≥ 99.5%, from Sigma-Aldrich.
All ICP-MS experiments during this study were performed using a Thermo Element 2 (Thermo Fisher Scientific, Bremen, Germany) mass spectrometric device, equipped with a double focusing (DF) sector field mass analyser applying low resolution (m/Δm = 400) for iodine and medium resolution (m/Δm = 4000) for sulfur detection, respectively. The ICP-MS instrument was fitted with a concentric nebulizer and a Scott double-pass spray chamber. For the flow injection set-up, the solvent was pumped using a peristaltic pump (0.5 mL min−1) and the sample injection was conducted with a dual mode injection valve from Rheodyne, model 9725 (Cotati, California, USA), fitted with a 20 µL PEEK injection loop (Upchurch Scientific, Oak Harbour, Washington, USA). Characterization studies of the iodinated antibody were carried out by SEC using a dual-piston liquid chromatographic pump (Shimadzu LC-10 AD, Shimadzu Corporation, Kyoto, Japan) equipped with a sample injection valve (Rheodyne, model 7125), fitted with a 50 µL injection loop, and a size exclusion Superdex 200 10/300 GL separation column (300 mm x 10 mm i.d., GE Healthcare Bio-Sciences, Sweden) having a fractionation range from 10 to 600 kDa. UV/VIS detection was performed with a Diode Array Detector (DAD) from Agilent Technologies (1100 Series, Waldronn, Germany)
2.3. Cell cultures and lysis
Human breast cancer cell lines MCF-7 and MDA-MB-231 were kindly provided by J. M. Pérez Freije (Dpto. of Biochemistry and Molecular Biology, University of Oviedo). These cell lines were cultured in 25 cm2 (T25) corning flasks (Sigma-Aldrich) with Dulbecco's Modified Eagle Medium (DMEM, LabClinics, Barcelona, Spain) sup-plemented with 10% Fetal Bovine Serum (FBS, Gibco, Life technologies, Madrid, Spain) and 5 µg mL−1 plasmocin prophylactic (InvivoGen, Nucliber, Madrid, Spain), and maintained in a humidified atmosphere of 5% CO2 at 37 °C. Six independent cultures were prepared for the MCF-7 cell line and five independent cultures were prepared for the MDA-MB-231 cell line. Confluent cells (80%) from each cell culture were washed with PBS (three times) and harvested by incubation with 2 mL of trypsin-EDTA solution (0.05% trypsin, 0.02% EDTA, Sigma-Aldrich) at 37 °C for 3 min. Following the trypsin treatment, the cells were collected in 10 mL DMEM and pelleted by centrifugation (200×g, 5 min). After removing the supernatant the pellets were washed three times with PBS and suspended in 1 mL of PBS. An aliquot of 20 µL of the cell suspensions from each replicate was used for cell counting using a hemocytometer. Moreover, in order to refer the quantitative results to the wet weight of the cells, the PBS of the cell suspensions was removed by centrifugation and the cells pellet in every replicate were weighed in a precision ( ± 0.01 mg) balance. After that, they were lysed by addi-tion of 1 mL of ice-cold water followed by three cycles of freezing/ thawing using liquid nitrogen. Finally, the cellular lysate was cen-trifuged (3000×g, 5 min) and the supernatant collected for further analysis. A flowchart of the above described experimental procedure is shown in Fig. 1.
2.4. Iodination of the anti-transferrin antibody
Iodination of the anti-transferrin antibody (anti-human transferrin polyclonal antibody T6265, Sigma-Aldrich) was carried using the Thermo Scientific™ Pierce™ iodination (Fisher Scientific) beads fol-lowing the advised protocol provided by the manufacturer with slight
Fig. 1. Flowchart of the experimental procedure used for cells culture and lysis.
modifications. Briefly, one bead was washed with 500 µL of Tris-HCl (0.1 M, pH 7) buffer during 30 s and dried on filter paper. Afterwards, this bead was loaded into a polypropylene microcentrifuge tube con-taining 250 µL of 10 mM NaI in Tris-HCl (0.1 M, pH 7) buffer and in-cubated for 5 min at room temperature. The production of free iodine could easily be observed by the change of the colourless solution to brown in less than 15 s. This part of the experiment was performed in a fume hood due to the presence of volatile iodine. In the next step 120 µL of the anti-transferrin antibody solution (1 mg mL−1 in 0.1 M Tris-HCl buffer, pH 7) were added to the reaction tube and the reaction was allowed to proceed for 4 min on ice. After this time, the reaction was stopped by removing the solution from the iodo-bead. Then, the iodi-nated-antibody was purified by passing this solution through a 30 kDa Amicon Ultra-0,5 Centrifugal filter (Merck Millipore, Darmstadt, Germany), the retentate fraction containing the iodinated antibody was washed three times with 0,5 mL of Tris-HCl (0.1 M, pH 7) buffer. Finally, the molecular weight cut-off filter was spun reversely at 1000×g to recover the iodinated antibody which was subsequently transferred into a polypropylene micro-vial and stored at 4 °C in Tris-HCl (0.1 M, pH 7) buffer for further use.