PIF4 | Phytochrome interacting factor 4 (goat antibody)
AS16 3955 | Clonality: Polyclonal | Host: Goat | Reactivity: Arabidopsis thaliana
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30 ul of total protein from Arabidopsis thaliana grown in 12 hour light/ 12 hour dark growth conditions and 6-day-old seedling samples taken at the end of the day (ZT12) were extracted with 100 mM MOPS, pH 7.6, 100 mM NaCl, 10% glycerol, 40 mM 2-mercaptoethanol, 5% SDS, 1X protease inhibitor cocktail from Roche, 2 mM PMSF. 80 µl buffer were added to about 100 µl grinded powder, then immediately heated at 70ºC for 10 minutes and separated on 8 % Bis-Tris SDS-PAGE and blotted 1h to PVDF using semi-dry or tank transfer. Blots were blocked with 5 % non-fat milk in TBST for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for over night at 4ªC with agitation in TBS-T. 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-goat IgG horse radish peroxidase conjugated) diluted to 1:10 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 minutes with chemiluminescent detection reagent. Exposure time was 1 minute.
Courtesy of Dr. Bo Zhang, Umeå Plant Science Centre, Sweden
PIF proteins are not that stable, therefore special precautions should be taken during extraction and whole procedure should be performed in as little light as possible (light green light).
Extraction of PIF proteins is described in Shen et al. (2007).