PetD | Cytochrome b6-f complex subunit 4

5 µg/well of total proteins were loaded/well. Proteins were extracted freshly from 10 ml of exponentially growing cyanobacterial cultures (0.5-1 OD730). Lysis was performed via beadbeating (0.1 mm glass beads) 3 times (20s 6000 rpm and 2 min on ice) in precooled 100 mM Tris-HCl (pH 7.6) 10% glycerol and cOmpleteTM, EDTA-free protease Inhibitor Cocktail (Merck). Then extracts were centrifuged at 17.000g for 25 min at 4°C to retrieve cytosolic fraction. Protein concentration was quantified using BCA assay and a BSA standard. Samples were denatured in 50 mM Tris-HCl (pH 6.8), 100 mM DTT, 2% SDS, 5 mM EDTA and 10% glycerol (final concentrations) + bromophenol blue stain. Denatured for 10 min at 95°C and separated in a 8-16% gradient SDS-PAGE using MOPS buffer. Samples were blotted in a Nitrocellulose membrane in a wet transfer system during 1.5h at constant current of 150 mA. Membrane was blocked overnight in 5% milk PBS-Tween buffer in agitation. Then, incubation of primary antibody (1:1000) performed during 1h at RT in 2.5% milk PBS-T. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed 3 times for 15 min in PBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:25 000 for 1h/RT with agitation in 2.5% milk PBS-T. The blot was washed as above and adding a last step with only PSB (no Tween added). And developed for 5 min with Agrisera AgriseraECLSuperBright with a LI-COR Odissey Fx Imager. Exposure time was 300 seconds.

Samples:
1 - 10 ug of 4 days old of wild-type Arabidopsis thaliana seedlings extract
2 - 5 ug of 4 days old of wild-type Arabidopsis thaliana seedlings extract
3- 10 ug of 4 days old of gun5-1 mutant seedlings extract

Exact buffer components were: (50 mM Tris–HCl pH 7.5, 10% glycerol, 150 mM NaCl, 10 mM MgCl₂, 5 mM EDTA, 5 mM DTT, 0.5% (v/v) Triton X-100, and 1 × protease inhibitors) and denatured with 4X SDS sample loading buffer (200 mM Tris-Cl (pH 6.8). 8% SDS (sodium dodecyl sulfate). 0.4% Bromophenol blue. 40% glycerol) at 95 °C 10 min.  Samples were separated in the RT on 15 % SDS-PAGE and blotted for 0.5 h to PVDF (pore size of 0.2 um), using:  semi-dry at room temperature. Blot was blocked with 5 % milk for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:  2 000 at 4°C with agitation overnight. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602 Agrisera) diluted to 1: 50 000 for 2 h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: AS16 ECL-N-10 AgriseraBright (mid picogram). Exposure time was 1 minute. Ther high cross reactivity above 50 kDa can be optimized by increasing blocking to 10 % nonfat milk and incubation of a primary antibody at 1: 2000 1h/RT. 

Courtesy of Dr. Duorong Xu, LMU München, Germany