cytc1 and cytc2 from following species: A. theoprasi, Brassica napus, Brassica oleracea, Cannabis sativa, C. maxima, Chlamydomonas reinhardtii (peptide target partially conserved), Lupinus luteus,  Medicago truncatula, Nicotiana tabacum, Oryza sativa, Ostreococcus (peptide target partially conserved), P. aurea, Physcomitrium patens, Ricinus communis, S. nigra, Solanum lycopersivum, Vigna radiata, Vitis vinifera.

Mitochondrial proteins (15 ug) from Arabidopsis thaliana mitochondria was separated on 16% acrilamide gel and electrophoresis prepared according to Schägger and von Jagov (Anl. Biochem., 1987, 166:368-379). After running the gel, proteins were transferred to PVDF membrane using wet transfer (Roti®-Blot 2, Roth). Transfer was checked by Ponceau S staining. Blot was destained by several quick washings in distilled water and 1 washing in 1X TBS (10 mM T pH 7.5, 150 mM NaCl) (10-15 min.).Blot was blocked by 1.5 hour in 5% milk in TBST (1X TBS, 0,1% Tween 20) After blocking blot was washed quickly twice in TBST and incubated 2 hours with primary antibody (dilution 1: 1000) in TBST. Washing: two quick washings in TBST and 3 x 10 min. washings in TBST. Then blot was incubated 45-60 min. with a secondary anti-rabbit antibodies conjugated to peroxidase (Agrisera AB, dilution 1:10 000, AS09 602) in TBST. Washing: as above. After washing blot was incubated 1-2 min. in chemiluminescent detection reagent. Chemiluminescence was detected by BioSpectrum® Imaging System (UVP). Exposure time was 5 seconds.

Courtesy Dr. Janusz Piechota, Wrocław University, Poland

Lane:

1 - MW marker

2 - 10 ug of Arabidopsis thaliana whole leaf extract

3 - 10 ug of Zea mays whole leaf extract

4- 10 ug of Solanum lycopersicum whole leaf extract

10 µg/well of total protein extracted from Arabidopsis thaliana, Zea mays and Solanum lycopersicum, total cell extract, stored at -80°C. Exact buffer components were 1xPEB (AS08 300) with protease inhibitor (0.1 mg/ml for Pefabloc SC, Roche) and denatured with Invitrogen LDS sample buffer (4X)  at 70°C/5 min. Samples were separated on Invitrogen NuPage Bis-Tris 4-12% SDS-PAGE  and blotted for 1 h to Invitrogen PVDF (pore size of  0.2  um), using wet transfer. Blot was blocked with 5% milk in TBS-T for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 500 for 1h/RT with TBS-T Blocking. 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 ALP conjugated, AS09 607 lot 2302 ) diluted to 1: 1 000 in TBS-T Blocking for 0,5h/RT with agitation. The blot was washed as above and developed with AS19 BCIP-NBT-PLUS lot 09269221 for 3min. As soon as the desired band was detectable, the membrane was washed in generous amounts of deionized water before placing the membrane on Whatman paper to dry. Image was captured after 1 h.

Courtesy Agrisera

Cytochrome c is located in inner mitochondrial membrane. It is a small heme protein which, unlike other cytochromes, is highly soluble. This protein is an essential component of the electron transport chain, where it undergoes oxidation and reduction without binding oxygen.