| | Re: como marcar un anticuerpo
Conjugated FITC by my self realized : mix equal volumes of antibody anti-IgG from goat with a saturated (4.1 M) and filtrate solution of ammonium sulfate adjusted at pH 7.0 with NH4OH o H2SO4, and incubate for 16 hrs at 4 °C. Centrifuge at 10900 rpm at 4 °C for 10 minutes, and therefore resuspend precipitate in FITC buffer (dissolve 0.05 M of boric acid, 0.2 M of NaCl, and adjust at pH 9.2 with 1 M NaOH solution, store at room temperature, stable one week). For to eliminate ammonium free ions, dialize through Visking 20/32 tubes, against the same FITC-buffer for 3 days with change of the dializing solution every 24 h. Determine the content of protein with the BCA method, and therefore add 20 µl of FITC (dissolve 5 mg/ml of FITC in dimethyl sulfoxide just before use) for every mg of antibody, and incubate for 2 hrs at room temperature. Remove free FITC dializing by means Visking 20/32 tubes, against stabilizing buffer (0.1 M Tris-HCl at pH 7.4, 0.1 % NaN3, 0.2 M NaCl, and adjust at pH 7.4 with 1 M NaOH, stable 1 week at room temperature) for 3 days with a change of the stabilizing buffer every 24 h. Determine again the proteins content, with the BCA method, aliquot in small part, and store at -20 °C, stable 2 years.