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Fluorescein labeling of proteins (antibodies) (II)

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1. Prepare pure protein in a suitable buffer (e.g., PBS. Tris interferes with coupling). Change to 0.1 M carbonate buffer (pH 9.0) if necessary.

2. Adjust protein concentration to ~ 10 mg/ml.

3. Prepare FITC (fluorescein isothiocyanate) 10 mg/ml in anhydrous DMSO. Vortex.

4. Slowly add 75 l FITC per 1 ml of protein solution.

5. Couple for 90 minutes in the dark at room temperature or overnight in the dark at 4 C with gentle agitation.

6. Separate labeled protein from free Fluorescein by dialysis or gel filtration. Concentrate if necessary.

7. Centrifuge at the maximum speed for 10 minutes and filter through 0.22 m filter. Add sodium azide to 0.05% as preservative.

8. Determine concentration and F/P (Fluorescein-labeled/total protein) ratio by measuring absorbance at 280 nm and at 495 nm. For antibodies, use the following formulae to get approximate values:

Protein concentration (mg/ml) = (A280 - 0.35 x A495) / 1.4
F/P ratio = (3.3 x A495) / (A280 - 0.35 x A495)