Anti-PTOX | Plastid terminal oxidase

Samples:
1 - Chloroplast samples corresponding to 3.0 µg chlorophyll of Arabidopsis thaliana
2 – Thylakoid membrane samples corresponding to 3.0 µg chlorophyll of Arabidopsis thaliana
3 – Stromal fraction samples corresponding to 3.0 µg chlorophyll of Arabidopsis thaliana
Left panel: MW markers

Protein samples corresponding to 1.0 µg chlorophyll of chloroplasts extracted from Arabidopsis thaliana.  Chloroplast samples were isolated with 20 mM Tricine–NaOH, pH 8.4, containing 400 mM sorbitol, 5 mM MgCl₂, 5 mM MnCl₂, 2 mM EDTA, 10 mM NaHCO₃, 0.5% (w/v) bovine serum albumin (BSA) and 5 mM ascorbate: and denatured with exact buffer (62.5 mM Tris-HCl, pH 6.8, containing 2% (w/v) SDS, 10% (w/v) glycerol, 0.005% (w/v) bromophenol blue, and 50 mM dithiothreitol) at 95 °C/5 min.  Samples were separated at RT on 15% acrylamide gel and blotted for 0.5 h to PVDF (pore size of  0.2  µm), using: semi-dry transfer at RT. Blot was blocked with 5 % milk for: 1h/RT with agitation. The blot was incubated in the primary antibody at a dilution of 1:  2,000 for 1h/RT with agitation in TBS-T. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (AS09 602 anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 25,000 in  for 1h/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 3 minutes.

Note: The band at 50 kDa is a cross reactivity with Rubisco. 

Courtesy of Prof.Yuki Okegawa, Okayama University, Japan

Samples:
l1 - Chlamydomonas reinhardtii, wild type cc124 (mt -)
2 - Chlamydomonas reinhardtii ptox2.34 (mt -)
3 - Chlamydomonas reinhardtii wild type cc125 (mt +)
4 - Chlamydomonas reinhardtii  ptox2.24 (mt +)
1x10e6 cells per lane of total protein extracted with Chloroform/Methanol protein precipitation protocol (dx.doi.org/10.17504/protocols.io.icecate)  from Chlamydomonas reinhardtii. Exact buffer components were: Count Chlamydomonas reinhardtii cell density in TAP cultures (early log phase), spin down 15 ml culture (4000 g, 5 min, 22°C) and resuspend pellet in 1 ml supernatant. Use 200 µl resuspended cells for protein precipitation in 2 ml Eppendorf tubes at room temperature (dx.doi.org/10.17504/protocols.io.icecate). Pellets were dried from MeOH and resuspended in 1x SDS PAGE running buffer (50 mM Tris/HCl pH 6.8, 50 mM DTT, 50 mM Na₂CO₃, 2% (w/v) SDS, 12% (v/v) Glycerol, 0.01% (w/v) Bromphenolblau, 0.01% (w/v) Servablau G) so that 20 µl corresponds to 1x10e6 cells. Sample was denatured (20 min, 22 °C) and spun down before loading. 13% SDS-PAGE Laemmli 1 h to nitrocellulose (pore size of  0.45 µm) semi-dry at room temperature. Ponceau S staining of total proteins to check loading and transfer efficiency. Follow recommended protocol for the remaining procedure (use agitation throughout; no double rinsing but in between blocking & antibody changes include 3x washing steps with TBS-T, 5 min/RT). Blocking: 5% non-fat milk 1h/RT, primary anti-PTOX antibody (Product No AS23 4895, Lot 2309) 1:1000 in TBS-T ON/4C, secondary antibody (AS09 602-trial) 1:25000 1h/RT, detection first with Agrisera Bright AS16 ECL-N. Exposure time was 3 s. 

Courtesy of Dr. Felix Buchert, University of Munster, Germany