AS20 4403 Anti-12S seed storage protein CRC

Arabidopsis thaliana dry seed extract from wilde-type (1) and atvst1-1 mutant (deffective in storage protein transport (2)  (SDS-PAGE, stained gel)  Western blot of the same samples (3,4) which were separated on 15-20 % SDS-PAGE and blotted to PVDF membrane in wet system. 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: 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.
P12S is the precursor of 12S globulin. 12S-alpha is the alpha-subunit (proteolyticall form of 12S) of 12S globulin.

Major 12S seed storage protein CRC (globulin) is synthesized on the endoplasmic reticulum as precursor and then transported to storage vacuoles, where it is processed at a conserved Asn-Gly peptide bond by an asparaginyl endopeptidase to produce two mature polypeptides referred to as alpha and beta subunits that are joined together by a disulfide bond. Phosphorylated in seeds on some Tyr residues in response to abscisic acid (ABA). 12S CRC protein is cleaved to alpha and beta chains. 
Cellular localisation: vacuole.

Alternative protein names: Cruciferin 3, Cruciferin C Legumin-type globulin Cruciferin1, Legumin-type globulin storage protein CRU1.

1. Shirakawa et al. (2014). CONTINUOUS VASCULAR RING (COV1) is a trans-Golgi network-localized membrane protein required for Golgi morphology and vacuolar protein sorting. Plant Cell Physiol. 2014 Apr;55(4):764-72.doi: 10.1093/pcp/pct195. (Immunohistochemistry, Western blot)
2. Li et al. MAG2 and three MAG2-INTERACTING PROTEINs form an ER-localized complex to facilitate storage protein transport in Arabidopsis thaliana. Plant J. 2013 Dec;76(5):781-91.doi: 10.1111/tpj.12347. (Immunolocalisation by electron microscopy, Western blot)

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