BiP | Lumenal-binding protein (chicken antibody)
AS09 614 | clonality: polyclonal | host: hen | reactivity: Arabidopsis thaliana, Hordeum vulgare, Spinacia oleracea, Zea mays
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|Recommended dilution||1 : 50-1 : 1000 (IF), 1 : 2000 (WB)|
|Expected | apparent MW||
73.5 | 80 kDa
|Confirmed reactivity||Arabidopsis thaliana, Hordeum vulgare, Physcomitrella patens, Spinacia oleracea, Zea mays
|Predicted reactivity||Nicotiana tabacum, Oryza sativa, Physcomitrella patens, Piea sitchensis, Populus trichocarpa, Spinacia oleracea, Zea mays|
|Not reactive in||No confirmed exceptions from predicted reactivity are currently known.|
Protein or membrane sample should be treated at 70°C for 10 min before loading on the gel.
|Selected references||Bennett et al. (2014). Plasma Membrane-Targeted PIN Proteins Drive Shoot Development in a Moss. Curr Biol. 2014 Dec 1;24(23):2776-85. doi: 10.1016/j.cub.2014.09.054. Epub 2014 Nov 13.|
5 µg of total protein from A.thaliana (1), H. vulgare (2), Z.mays (3), S. oleracea (4), extracted with Agrisera PEB extraction buffer (AS08 300) were separated on 4-12% SDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 10 000 for 1h at room temperature 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 room temperature with agitation. Blots were incubated in secondary antibody (anti-hen IgY horse radish peroxidase conjugated, from Agrisera AS09 603) diluted to 1:50 000 for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL detection reagent according to the manufacturers instructions. Exposure time was 5 seconds.
BiP localization in 5 days old Arabidopsis thaliana roots. BiP signal shown in green, DAPI in blue. The material has been fixed in para-formaldehyde for 30 minutes. Tissue cleaning has been performed before immunolocalization. Chicken anti-BiP primary antibody was diluted in 1: 1000 and DyLight®488 conjugated goat anti-chicken secondary antibody AS09 622 (green color) was diluted in 1: 1000. Co-staining with DAPI visualized nucleus (blue color). Scale bar – 10 µm.
Courtesy Dr. Taras Pasternak, Freiburg University