PMC:2779105 / 189683-191774 JSONTXT

{"project":"2_test","denotations":[{"id":"19956594-19198665-88163131","span":{"begin":2088,"end":2089},"obj":"19198665"},{"id":"T30941","span":{"begin":2088,"end":2089},"obj":"19198665"}],"text":"Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)\nNB-like particles containing fetuin-A and/or albumin like the ones shown in Fig. 8 were prepared by diluting BSF (Sigma) at a final concentration of 20–160 µg/ml in DMEM or HSA (Talecris) at a final concentration of 0.2–1.6 mg/ml in DMEM. The precipitating reagents CaCl2 and NaH2PO4 were then added each at a final concentration of 3 mM. A final volume of 1 ml of DMEM was used. Incubation was done in cell culture conditions for 1 month. The particles were then pelleted by centrifugation at 16,000× g for 15 min at room temperature. The pellet was washed twice with HEPES buffer using the same centrifugation steps. The particles were resuspended in 50 µl of 50 mM EDTA. Each sample was mixed with the 5X “loading buffer” (0.313 M Tris-HCl pH 6.8, 10% SDS, 0.05% bromophenol blue, 50% glycerol, 12.5% β-mercaptoethanol) to obtain a final concentration of “loading buffer” of 1X in a volume of 20 µl. 50 mM EDTA was used to dilute the samples. The protein solutions were heated at 95°C for 5 min and were subsequently loaded on a 10% SDS-polyacrylamide gel. For Fig. 8A, BSF-NLP were prepared by using DMEM (final volume of 1 ml) containing BSF (Sigma) at 20 µg/ml (lane 1), 40 µg/ml (lane 2), 80 µg/ml (lane 3), and 160 µg/ml (lane 4), followed by addition of CaCl2 and NaH2PO4 each to 3 mM and incubation in cell culture conditions for 1 month. Following incubation, the particles were pelleted as described above and washed twice with DMEM. The pellet was resuspended in 50 µl of 50 mM EDTA and 4 µl of each sample was loaded in the lanes described above. For Fig. 8B, HSA-NLP were prepared in a similar manner by using DMEM containing HSA (Sigma) at 0.2 mg/ml (lane 1), 0.4 mg/ml (lane 2), 0.8 mg/ml (lane 3), and 1.6 mg/ml (lane 4). For Fig. 8C, BSF-HSA-NLP were prepared similarly by using DMEM containing both proteins at the concentrations mentioned above. Gel electrophoresis was performed using a mini-gel system (Hoefer, Holliston, MA, USA). The gels were stained with Coomassie blue as described earlier [2]."}