FITC labeling of p43

Before labeling

For conjugation of FITC to protein, protein concentration should be over 1mg/ml. If your protein concentration is below 1mg/ml, conjugation efficiency will be decreased.

Must be prepared

1. Protein

2. 500 mM carbonate buffer(pH 9.5) at 4 degree; 17g/L Na2CO3, 28g/L NaHCO3

3. Dissolve FITC in DMSO(2mg/ml), use fresh one.

4. 1 M Tris-HCl(pH 6.8)

Procedures

1.       p43 : 1.5mg/ml

2.       Dialyze against 500mM carbonate buffer with 2 changes at 4 degree

3.       Remove and place in a microfuge tube

4.       Add 100ul of FITC solution(200ug/mg protein); cover with foil to darken. Add  slowly with pippete-man, no more than 10% at a time.

5.       Place on rotor at 4 degree for 6 to 19 hrs. Alternatively, 2 hrs at RT will suffice.

6.       To stop reaction, centrifuge the tube at 2,000 rpm for 5 min

7.       Harvest supernatant and discard pellet.

8.       Add 100ul of 1M Tris-HCl(pH 6.8) to the supernatant and, mix well.

9.       Dialyze sample against PBS(pH 7.4) containing 20% glycerol with 5 changes over 40hrs.

10.  Harvest and spin in microfuge tube at 14,000rpm for 10 min.

11.  Store at 4 degree in the dark tube to protect from light.

12.  Determine F/P ration by reading A280 protein in mg/ml and A495.The F/P is then determined using :

                                       2.87 X A495

           Molar ration =  ——————-

                                    A280-0.35 X A495