* **FITC Labeling of Proteins**((Adapted from Dr. Janos Szollosi at Medical University School of Medicine, Debrecen, Hungary and MarkerGene<sup>TM</sup> FITC Antibody Labeling Kit M0955))
    * // The following protocol can be used to label proteins with concentrations 2-10mg/ml // 

    - Set the pH of the protein solution to 9.3 via dialysis against 2L of 100mM Sodium Carbonate at 4<sup>o</sup>C for 24 hrs
      *Change the dialysis solution once during this process
    - Measure OD<sub>280</sub> of protein solution 
      *Calculate Molar Concentration ([Protein] M) using OD<sub>280</sub>/extinction coefficient of Protein
    - Weigh out 3-4 mg of FITC and add it to 1 mL of 100 mM Sodium Carbonate pH 9.3
    - Dilute FITC solution 100x with 100mM Sodium Carbonate pH 9.3
      *Use stock and diluted FITC solution within 1-2 hrs
    - Measure OD<sub>495</sub> and OD<sub>280</sub> of dilute FITC solution. Record rFITC = OD<sub>495</sub>/OD<sub>280</sub>
      *rFITC should be between 3 and 3.3
    - Calculate Molar Concentration of FITC ([FITC] M) = OD<sub>495</sub>/extinction coefficient at pH 9.3 (73,000)
    - Add 15-20ug of FITC per mg of protein. If [protein] is less than 5mg/ml, [FITC] should not be lower than 100ug/ml 
    - Incubate for 1 hr at room temperature or overnight at 4<sup>o</sup>C
    - To purify, use a Sephadex G25 Column primed with 1x PBS pH 7.4
    - The first eluent with a greenish tint should be collected and saved as this is the labeled protein
    - Calculate labeling ratio as follows:
      * [Protein] mg/ml = (OD<sub>280</sub> FITC-Protein - ((OD <sub>495</sub> FITC-Protein)/rFITC)/1.4
      * [Protein] M = ([Protein] mg/ml)/MW Protein
      * [FITC] M = (OD<sub>495</sub> FITC-Protein)/FITC coefficient of extinction at pH 7.4 (63,000)
      * Labeling Ratio F/P = ([FITC] M)/([Protein] M)
    - For Best Results F/P should be between 2 and 4, 1 and 5 are acceptable, but not ideal

----

Edited by Mitchell Weiser 3/1/07