1-15µg of chlorophyll from isolated chloroplasts from Arabidopsis thaliana, extracted with a buffer containing (25 mM Tricine-NaOH, pH 7.8, 330 mM sorbitol, 1 mM EDTA, 10 mM KCl, 0.15% [w/v] bovine serum albumin, 4 mM sodium ascorbate, and 7 mM L-Cys) were separated on 12 % SDS-PAGE and blotted 1h to PVDF using semi-dry transfer. Blots were blocked with 10% milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 overnight at 4⁰C 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 secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602) diluted to 1:10 000 in TBS-T for 1h at RT with agitation. The blot was washed as above and developed for 60 seconds with a ImageQuant system from GE Healthcare, exposure time was 60 seconds.

Courtesy of Dr. Rikard Fristedt, University of Amsterdam, The Netherlands

RAF2 (Rubisco accumulation factor 2) is a member if PCD family and a  chloroplastsic protein which cotains pterin carbinolamine dehydratase domain. Protein is involved in tetrahydrobiopterin biosynthetic process.

Alternative names: AT5g51110/MWD22_5, PCD/DCoH-like protein 1, Transcriptional coactivator/pterin dehydrataseImported.