C/EBPα Blot

• 1. Run gel @ 100-125V for 90-120 minutes until dye front has reached the bottom

1. Transfer to shiny side of PVDF membrane

a. 100 V for 90 minutes using ice

b. 30 V overnight

1. Block membrane with 5% milk in TTBS for at least 2 hours @ room temp, or overnight at 4 ̊C on the rotator.

2. Briefly rinse the blot with TTBS

3. Dilute Int. C/EBPα A.P. Ab 1:1000 in TTBS with 1% γ-globulins. Shake membrane with Ab. @ room temp for 2 hours.

4. Wash blot in ~20 ml volumes of TTBS 3 times for 5 minutes, and once for 15 minutes.

5. Prepare secondary Ab α-rabbit HRP dilute 1:4000 in TTBS with 5% milk, shake blot with secondary Ab 1 hour at room temperature.

6. Repeat washing (step 6)

7. Mix the ECL using 3 mL each of the Pico and 300 μL each of the Femto, incubate the blot in ECL for ~5 minutes.

8. Wrap blot in plastic and expose to the film.