Sephadex g 25 column

The homogeneous mono [¹²⁵I A14] Insulin used as a tracer for both radiometric methods, RBA and RIA, was produced using recombinant human insulin (HumulinC from Eli Lilly, Indiana, USA) devoid of preservative substances by chromatography on a Sephadex G25 column (Amersham Biosciences, New Jersey, USA). The labeling and HPLC purification steps were carried out following as indicated by Linde et al. [3] (link) and Jørgensen et al. [4] , respectively. Specific radioactivity of roughly 300–380 μCi/μg was routinely achieved.
The concentration of tracer in both, RBA and RIA, was 5.8 × 10⁻¹¹, calculated from an activity of 20,000 cpm; Relative mass of insulin, 5808 Da; Final incubation volume, 0,12 mL; Gamma counter efficiency, 70%; Specific activity of fresh insulin tracer, 380 μCi/μg.

For SEC separation, 5 g of hydrolyzed protein was mixed with 35 mL of HCl (0.01 N). Then, three volumes of ethanol were added to this solution and stored at 4 °C during the night for deproteinization. The mixture was centrifuged at 12,000 rpm for 20 min at 4 °C to remove the precipitated proteins, and ethanol was evaporated using a rotary evaporator. Samples were lyophilized overnight (SCANVAC, Labogene ApS, Denmark). Before injection into the SEC column, 1 g of the dried sample was resuspended in 10 mL of 0.01 N HCl and filtered through a 0.45 μm syringe filter to remove non-soluble impurities. A total of 5 mL of the sample was injected on the Sephadex G25 column (2.5 × 65 cm) (Amersham Biosciences, Uppsala, Sweden). The separation process was performed using a flow rate of 15 mL per hour of degassed 0.01 N HCl, and 5 mL peptide fractions were collected using an automatic collector. Finally, the absorbance was measured at 254 and 280 nm in a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Palo Alto, CA, USA), and the biological activities were also assayed in all fractions [24 (link),25 (link)].

After each treatment, the incubation medium was removed and cells were washed with PBS and lysed with 200 μL of 0.1 M potassium phosphate buffer pH 7.5 which contained 0.5% Triton X-100. The lysed cells were centrifuged at 13,000 rpm for 5 min before collecting supernatants. Reduced cytochrome c (7 mg) was dissolved in 1 mL of 0.1 M of potassium phosphate buffer pH 7.5 and dithionite was added to ensure cytochrome reduction. Excess dithionite was removed by passing the cytochrome c solution through a Sephadex G-25 column equilibrated with the buffer (Amersham Bioscience, Arlington, MA, USA). The eluyent was taken and adjusted to an optic density of 0.7 absorbance. The extract was added to 1 mL of this solution and absorbance measured at 550 nm. Activity was expressed as OD/10 min/mg protein.