TGG1 | Myrosinase 1 ( BGL38)
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Arabidopsis thaliana total leaf was freshly extracted with 2x SDS-sample buffer (+ 2ME) for SDS-PAGE and denatured with 4X SDS buffer at 95°C for 5 min. 10 µg of protein was loaded and separated on 15-20 % SDS-PAGE and blotted 1h to PVDF membrane. Blot was blocked with 3 % skim milk/TBS-T, 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 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.
Immunolocalization of TGG1 in sections of Arabidopsis thaliana 48 days-old rosette leaves. Panel to the left: CBB staining (a,c). Middle panel: Reaction with anti-TGG1 antibodies used at 1: 1000 dilution and followed by visualization with AlexaFluor488 goat anti-rabbit antibodies at 1: 1000 (b.d). Right panel: electron microscopy of ultrathin sections mounted on Formvar-coated nickle grid. Anti-TGG1 antibodies were used at 1: 1000 dilution and following washing in PBS the sections were incubated with anti-rabbit IgG conjugated gold particles (AuroProbe EM). CW- cell wall, V- vacuole.
Rosette leaves of Arabidopsis thaliana were fixed with 4% (w/v) paraformaldehyde and 1% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) at 4°C for 3 h. After washing with 0.02 M cacodylate buffer (pH 7.4), these tissues were dehydrated with acetone and embedded in LR white resin at –20°C. Sections were cut on an ultramicrotome (Leica, Reichert Division, Vienna, Austria) for both light microscopic and electron microscopic analyses.
Shirakawa et al. (2010). Arabidopsis Qa-SNARE SYP2 proteins localized to different subcellular regions function redundantly in vacuolar protein sorting and plant development. Plant J. 2010 Dec;64(6):924-35.doi: 10.1111/j.1365-313X.2010.04394.x. (Western blot)
Ueda et al. (2006). AtVAM3 is required for normal specification of idioblasts, myrosin cells. Plant Cell Physiol. 2006 Jan;47(1):164-75. doi: 10.1093/pcp/pci232. (Immunlocalization, Western blot).
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