Erlenmeyer flask with 50 mL P. tricornutum culture was coated with aluminium foil to prevent induction of P_fcpB.

To demonstrate the production of antibodies in the induced cultures 2.1 and 4.2 a Western Blot should be performed. For this purpose, the cells were harvested and the pellet was separated from the supernatant. After that, a TCA precipitation was performed. All samples were stored at -20°C.

After two days of incubation in darkness, the culture was divided in 5 samples, which were incubated under 5 different light conditions: normal light, green light, red light, blue light (50 µE each) and darkness. The division was realised in darkness to prevent induction of the light-inducible promoter. OD (600 nm): 0.450.

The samples that were taken the previous day and the days before induction with nitrate were used for a 12 % SDS gel (see above) and subsequent Western blotting on a nitro cellulose membrane. To accomplish this four Whatman filter papers were soaked into membrane buffer and a nitro cellulose membrane, also soaked in membrane buffer, were stacked. On this the SDS gel was placed and covered with with four Whatman filter papers soaked with gel buffer. Then the proteins were transferred with 0.8 mA/cm² for two hours.

To prevent unspecific binding of the first antibody, the membrane was rotated for at least one hour in 5 % milk powder in TBST at 4 °C. The primary antibody was dissolved in 2 % milk powder in TBST (dillution: 1:500 anti GFP, anti IgG 1:10,000, all antibodies obtained by Santa Cruz biotechnology) and added to the membrane. Afterwards it was washed two times in TBST for five minutes. The secondary antibody was dissolved in 2 % milk powder in TBST (anti anti GFP 1:10,000, anti anti IgG 1:10,000), added to the membrane and incubated for one hour. Afterwards it was washed with TBST for three times for five minutes each. After incubation with a detection agent, containing Luminol, p-Coumaric acid and H2O2, the detection was performed with a Chemocam Professional from INTAS science imaging.

Sample preparation:
• Resuspend pellet in 1 ml water
• Measure OD (600 nm)
• Take the volume corresponding to a certain OD
• Protein preparation of the taken sample with the predefined OD
  • Cell culture is centrifuged for 5 minutes (full speed)
  • Discard supernatant
  • Add 150 µl sodium hydroxide
  • Incubate 10 minutes on ice
  • Add 850 µl bidest. water
  • Add 200 µl 70 % TCA and incubate for 20 min on ice
  • Centrifuge for 15 minutes at 4 °C (20000xg)
  • Wash pellet with aceton and centrifuge for 15 minutes at 4 °C
  • Repeat the latter step until the chlorophyll is washed out
  • Dry pellet at room temperature
  • Resuspend in 8 M urea buffer
  • Shake the samples for 15 min at 60 °C
  • Load samples on a 12 % SDS gel
• Load samples on the gel
• Run gel for at least 1 h (120V)
  • Mini-Protean Tetra Cell (Biorad)
• Blotting on a nitrocellulose membrane over night (20 V)
• Submerge blot in 4 % milk powder in TBST
• Incubate for 2 h at 4 °C
• Incubate membrane for 1-2 h with the milk and the primary antibody against GFP and tubulin (dilution: 1:1000 GFP, 1:2000 tubulin)
• Wash membrane in TBST for 10 min (3 times)
• Incubate for 1 h at 4 °C with the secondary antibody in milk-TBST (goat anti mouse HRP)
• Wash for 10 min at 4 °C in TBST (3 times)
• Detection in western imager station (chemocam)
  • ECL solution: incubate membrane for 1 min in ECL solution