Labeling of Immunoglobulins with Fluorescent Dyes

Add 33 pi of fluorescein isothiocyanate (FITC tetramefliylrho-damine isothiocyanate, TRITC, or another fluorescent dye isothiocyanate derivative is used the same way), 50 mg/ml in DMF, to 1 ml of 5 mg/ml IgG in Soln. A. Shake at RT protected from light for 1 h. Remove surplus FITC and its hydrolysis products on a Sephadex G-25 column, equilibrated with PBS. The conjugate appears in the void volume and should be concentrated by ultrafiltration. Add sodium azide to a final concentration of 0.02% (w/v) and glycerol up to 10% (w/v) and store at 4 °C. 

Calculate the degree of substitution F/P (nmoles FITC per nmoles IgG) using the following equation  

absorption of the sample at 495 nm and 1 cm path V, total volume of the conjugate in milliliters P, total amount of IgG in micrograms. 

The protein concentration P is determined by measuring the sample dissolved in PBS (reference PBS) at 280 nm with consideration to the fluorochrome-specific absorption at 495 nm  

The ratio F/P (moles fluorochrome per milligram of protein) is calculated by 

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