Purification of APP C100-FLAG substrate

Purification of APP C100-FLAG substrate

1. Incubate 50 µl BL21(DE3)-competent bacteria with 1 µl plasmid (C100-FLAG in pET22b, 10 ng - 100 ng) for 30 minutes on ice. Heat shock using the hot plate for 45 seconds at 42°C. Incubate on ice for 2 mins. Add 400 µl LB medium warmed to 37°C, mix, and incubate at 37°C for 60 min. 

2. Spread the transformation reaction on LB agar plates supplemented with 100 µg/ml ampicillin. Incubate at 37°C overnight.

3. Inoculate 100 ml of LB medium supplemented with 100 µg/ml ampicillin in a 500 ml flask, with a single colony from the plate. Grow overnight at 37°C with agitation at 200 rpm in an incubated shaker. 

4. Add 5 ml of the 100 ml overnight culture (pre-culture) to 500 ml (in a 2 liter flask) of Terrific broth supplemented with 100 µg/ml ampicillin (use as many flasks as desired depending on the amount of protein needed). Grow at 37°C with agitation at 250 rpm until the OD600 reaches 0.8. Keep 1 ml of culture as control expression before induction. 

5. Add IPTG to a final concentration 1 mM and incubate for 3 h at 37°C with agitation at 250 rpm. 

6. Cells are then pelleted at 8,000 g for 20 min.

7. Re-suspend cell pellets in lysis buffer containing 50 mM HEPES, pH 7.0, 1% Triton X-100 detergent supplemented with protease inhibitor cocktail. Cells are lysed by French press. 

8. Centrifuge the solution at 100,000 g to obtain clear lysate.

9. Isolate FLAG-tagged APP-C99 immunoprecipitation for 3 h at 4°C with anti-FLAG M2 beads from Sigma. 

10. Elute the FLAG-tagged APP-C99 with 100 mM glycine, pH 2.5, 0.25% NP40 prior to being neutralized with Tris buffer and stored at -80 ̊C.