SMT1 | Sterol methyltransferase 1
AS07 266 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana | Marker antibody of integral ER membrane
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Hordeum vulgare, Triticum aestivum, Withania somnifera
Total protein from Col-0 (wild-type) Arabidopsis thaliana were extracted with 50mM HEPES-KOH buffer containing 250 mM sucrose, 5% glycerol, 50 mM NaPP, 1 mM NaMo, 25 mM NaF, 10mM EDTA, 0.5% PVP, 3mM DTT, 1mM PMSF, 10uM Leupeptin & 10nM Calyculin, and then fractionated by ultracentrifugation at 100,000 x gravity for 30 min at 4°C into soluble (S100) and microsomal (P100) proteins as described in LaMontagne et al. (2016). Isolation of Microsomal Membrane Proteins from Arabidopsis thaliana. Current Protocols in Plant Biology 1:1-18. doi: 10.1002/cppb.20020. 30 µg proteins of total, S100 and P100 fractions were denatured at 37°C for 5 min, separated on a 7.5 % SDS-PAGE and blotted 1h to nitrocellulose using tank transfer. Blots were blocked with 1x PBS (from Fisher Scientific BP665-1) + 0.1 %Tween 20 (PBS-T) + 5% milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 overnight at 4°C with agitation in 1x PBS-T + 5% milk. The antibody solution was decanted and the blot was rinsed briefly once, then washed four times for 7 min in 1x PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in 1x PBS-T + 5% milk for 2 hrs at RT with agitation. The blot was washed as above and developed for 4 min with chemiluminescent detection reagent. Exposure time was 30 seconds and 2 min.
SMT1 is an integral membrane protein of ER (Boutte et al., 2009) while BiP is a membrane associated protein.
Endogenous SMT1 is expressed at very low levels in root epidermis and root cap as compare to cortex and endodermis and therefore this can contribute to SMT1 detection problems.
SMT1 (EC=126.96.36.199) is an enzyme involved in steroid biosynthesis localized specific to the Endoplasmic Reticulum (ER), Boutte et al., 2009 . It is catalyzing the methyl transfer from S-adenosyl-methionine to the C-24 of cycloartenol to form 24-methylene cycloartenol. The protein is highly expressed in vascular tissue, mature leaves and in regions undergoing cellular expansion. Alternative names: Cycloartenol-C-24-methyltransferase, 24-sterol C-methyltransferase 1, Protein STEROL METHYLTRANSFERASE 1, Protein CEPHALOPOD
LaMontagne et al. (2016). Isolation of Microsomal Membrane Proteins from Arabidopsis thaliana. Curr. Protoc. Plant Biol. 1:217-234. doi: 10.1002/cppb.20020.
Yoshimoto et al. (2014). Quality control of plant peroxisomes in organ specific manner via autophagy. J cell Science, August 1, 2014, 127 (15).