CPX1 | Coproporphyrinogen-III oxidase 1

KLH-conjugated synthetic peptide derived from Arabidopsis thaliana CPX1 sequence, UniProt:
UniProt: Q9LR75,  TAIR: AT1G03475

1 : 2000 (WB)

Samples:
1: Cucurbita pepo sbsp pepo
2: Cucurbita pepo sbsp pepo
3: Cucurbita pepo sbsp pepo
4: Cucurbita pepo sbsp pepo
5: Arabidopsis thaliana Col-0

5 µg/well of total protein extracted freshly from Cucurbita pepo or Arabidopsis thaliana leaves were extracted with (0.2M NaCl, 0.02M Tris-HCl pH 7.5, 1mM EDTA, 1mM PMSF, 1% NP-40, 0.2% SDS and denatured with Laemmli sample buffer at 95°C for 4 min. Proteins were separated on 8 % SDS-PAGE  and blotted 1h to PVDF (pore size of  45 um), using semi-dry transfer. Blot was blocked with 5 % milk for 1h/RT. Blot was incubated in the primary antibody at a dilution of 1: 2 000 in TBS-T for ON/4°C with agitation. 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 Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1: 5 000 in TBS-T for 1h/RT with agitation. The blot was washed as above and developed for 2  min with Agrisera ECLBright. Exposure time was 2 min.

Courtesy of Dr.Theoni Margaritopoulou,Benaki Phytopathological Institute, Greece