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C/EBPα Blot

  1. Run gel @ 100-125V for 90-120 minutes until dye front has reached the bottom
  2. 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.