PYK10 | Beta-Galactosidase (internal) (ER marker, immunolocalisation)
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Arabidopsis thaliana 7 day-old seedlings was freshly extracted with 2x SDS-sample buffer (+ 2ME) for SDS-PAGE and denatured with 4X SDS buffer at 95°C for 5 min. were separated on 12.5 % SDS-PAGE and blotted to PVDF membrane in semi-dry system. Blot was blocked with 5 % skim milk/TBS-T, 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5000 in TBS-T for 1h/RT. The antibody solution was decanted and the blot was washed 4 times for 10 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in for 1h/RT with agitation. The blot was washed as above and developed with a chemiluminescent detection reagent, following manufacture's recommendations.
Left panel: anti-PYK10 antibodies, middle panel: GFP, right panel: marged
The P1 pellet was obtained from 8 day-old seedlings (carrying GFP gene with ER-retension signal). Anti-PYK10 antibodies: 1: 500, secondary antibodies: goat anti-rabbit IgG Alexa Fluor546 at 1: 1000.
PYK10 is localising to ER bodies,
Nagano et al. (2005). Activation of an ER-body-localized ß-Glucosidase via a Cytosolic Binding Partner in Damaged Tissues of Arabidopsis thaliana. Plant Cell Physiol. 2005 Jul;46(7):1140-8. doi: 10.1093/pcp/pci126. (Immunoprecipiation, Western blot, Arabidopsis thaliana)
Matshushima et al. (2003). A novel ER-derived compartment, the ER body, selectively accumulates a beta-glucosidase with an ER-retention signal in Arabidopsis. Plant J . 2003 Feb;33(3):493-502. doi: 10.1046/j.1365-313x.2003.01636.x. Immunofluorescence, Immunohistochemistry, Western blot, Arabidopsis thaliana)
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