PIF3 | Phytochrome interacting factor 3 (goat antibody)
AS16 3954 | Clonality: Polyclonal | Host: Goat | Reactivity: Arabidopsis thaliana
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Total protein from Arabidopsis thaliana (10-20 µg) of 4-d-old dark grown Col-0, pif single, triple and quadruple mutants were extracted with the buffer described in Qiu et al. 2015 and denatured at 95°C for 5 min. were separated on 8 % Bis-Tris SDS-PAGE, and subsequently blotted to Nitrocellulose membrane using wet tank transfer. Blots were blocked with TBS containing 2% non-fat milk at room temperature for 1h. Primary anti-PIF3 antibodies were used in dilution of 1:1000 to TBS containing 2% milk. The incubation time was 14-20 hours at 4°C. After washing 4x5 min. with TBST at room temperature, the blots were incubated with HRP-conjugated secondary antibodies (1:5000) at room temperature for 1h. The blots were washed 4x10 min with TBST again and the signal was detected using the SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific).
Courtesy od Dr. Yongjian Qiu, University of California-Riverside, USA
Please, do not re-use PIF3 antibody solution after first incubation with your membrane as most of antibody will bind in this step and next result will not be reproducable.
PIF3 protein runs at higher MW than expected, as observed previously (Al-Sady et al. 2006). 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).
Sinclair et al. (2017) Etiolated Seedling Development Requires Repression of Photomorphogenesis by a Small Cell-Wall-Derived Dark Signal. Curr Biol. 2017 Nov 20;27(22):3403-3418.e7. doi: 10.1016/j.cub.2017.09.063.
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