Author: Unkown. Date: 1/11/96

Standard Operating Procedure:

FITC (Cellite) Conjugation

Hazards:        Fitc-Cellite is an irritant, toxic at high levels

 

Protection:    wear gloves

 

Waste:           

 

Spill Clean-Up:

 

Procedure:

 

Prepare bicarb buffer:

            make up:       .5M NaHCO3            (42g in 1L)    

                                    .5M Na2CO3             (10.6 g in 200 ml)

            add carbonate to bicarbonate until desired pH is achieved

 

1. Wash a desalting column (Pierce #20449, Kwiksep) with 15-30 ml PBS.

 

2. Meanwhile, adjust pH of antibody solution to 9.2 (Use 1/10 volume bicarb buffer if the antibody is in PBS).

            (Other protocols recommend pH 8.5, but I’ve found the conjugation is MUCH better at higher pH. Some antibodies may not withstand this, however. Be sure to minimize the time the antibody is at this pH)

 

3. Add 2.5 mg of FITC Cellite (Calbiochem #34323, 1g) to antibody solution.

            Turn by hand for 3 minutes.

 

4. Centrifuge at setting “5” for 3 minutes.

 

5. Apply the supernatant to the column, allow to flow down.

            Continue to apply PBS, 1ml at a time.

            Collect 10 1ml fractions.

            With this column, the antibody is usually in fractions 2-5.       

 

6. Test fractions for antibody by staining control cells.

 

7. Pool positive fractions, determine final concentration to use at.