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.

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)
• Equilibrate the gel in Towbin transfer buffer (25 mM Tris, 192 mM glycine pH 8.3, 20 % MeOH) for 10 min
• Blotting on a nitrocellulose membrane using the STANDARD SD transfer protocol from Biorad preprogrammed protocols (Trans-Blot Turbo Blotting System)
• 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

10 mL of the culture were harvested for cell disruption with sonication (see protocol below), 1 mL for cell disruption with the ball mill, and 1 mL as a reserve. Cells were disrupted with sonication and the ball mill. Optical density (at 600 nm) was measured for each sample.

As a positive control, we extracted a protein-coding gene, cry-gfp, out of Arabidopsis landsberg erecta, which produces GFP as well as tubulin. For this purpose, 228 mg leafs were mixed with 114 µL PEG extraction buffer. Following this, the cells were disrupted with a Polytron PT 1200E Blade-type homogenizer (Brinkmann) and 5 min in a sonication bath. After centrifugation (15 min, 20000g, 4°C), the supernatant was used as protein extract. The concentration of protein was determined using a Bradford assay (Roti Quant, Roth).

Result: 5.16 µg/µl protein were extracted.

5 µg (ball mill) and 10 µg (sonication) protein was used for SDS-PAGE. After that, a Western Blot was performed overnight (4°C, 30V).

After blotting procedure, the following protocol was applied:
• Blocking: Incubate in 7 % milk powder in TBST buffer for 1 h at RT
• Wash three times for 10 min with TBST buffer at RT
• Add the first antibody: goat anti-GFP (1:2000) in TBS with 0.02% NaN3 for 1.5 h at RT
• Add the second antiboy: anti-goat 800 (1:10000) in TBS buffer for 1 h at RT
• Wash two times for 10 min with TBST buffer at RT
• Wash a last time for 10 min with TBS buffer at RT
• Detect with scanner at 800 nm
• Add the first antibody: mouse anti-tubulin (1:2000) in TBS with 0.02% NaN3 for 1.5 h at RT
• Add the second antiboy: anti-mouse 800 (1:10000) in TBS buffer for 1 h at RT
• Wash two times for 10 min with TBST buffer at RT
• Wash a last time for 10 min with TBS buffer at RT
• Detect with scanner at 800 nm

Western blot

Gel substances 12 % Acryl amide gel 6 µl PageRuler Prestained Protein Ladder (Thermo Scientific) Upper gel Lane   Content   Expectations 2   Darkness   27 kDa (GFP) + 55 kDa (tubulin) 3   Blue light   27 kDa (GFP) + 55 kDa (tubulin) 4   Green light   27 kDa (GFP) + 55 kDa (tubulin) 5   Red light   27 kDa (GFP) + 55 kDa (tubulin) 6   Control   55 kDa (tubulin) + 80 kDa ("GFP") Lower gel Lane   Content   Expectations 8   Light   27 kDa (GFP) + 55 kDa (tubulin) 9   Darkness   27 kDa (GFP) + 55 kDa (tubulin) 10   Blue light   27 kDa (GFP) + 55 kDa (tubulin) 11   Green light   27 kDa (GFP) + 55 kDa (tubulin) 12   Red light   27 kDa (GFP) + 55 kDa (tubulin) 13   Control   55 kDa (tubulin) + 80 kDa ("GFP")

Result: All samples showed the expexted bands. For a detailed discussion have a look at our light control section.