AS20 4425 Anti-ASN | Glutamine-dependent asparagine synthetase

Arabidopsis thaliana total leaf extract was freshly extracted with 2x SDS-sample buffer (+ 2ME) for SDS-PAGE and denatured with 4X SDS buffer at 95°C for 5 min. Protein was loaded/well and were separated on 10% 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 recommendation. Molecular weight of ASN2 is 65 kDa. ASN1 is expressed in floral organs, while ASN2 is expressed in leaf. 

Arabidopsis thaliana total leaf extract and respective mutants were 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/well abd samples were separated on 10% 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: 2500 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 recommendation.

For images of sections and IHC method protocol, please refer for listed publications. 

This antibody reacts with both isoforms: ASN1 and ASN2

ASN (Glutamine-dependent asparagine synthetase) is essential for nitrogen assimilation, distribution and remobilization within the plant via the phloem. ASN2 is expressed in leaf and ASN1 is expressed in floral organs. The amino acid sequences of Arabidopsis thaliana ASN1 and ASN2 are 76% identical. The amino acid sequences of Arabidopsis thaliana and Zea mays ASN2 are 73.6% identical.

1. Gaufichon et al. (2017). ASN1-encoded asparagine synthetase in floral organs contributes to nitrogen filling in Arabidopsis seeds. Plant J. 2017 Aug;91(3):371-393. doi: 10.1111/tpj.13567.
2. Gaufichon et al. (2013). Arabidopsis thaliana ASN2 encoding asparagine synthetase is involved in the control of nitrogen assimilation and export during vegetative growth. Plant Cell Environ. 2013 Feb;36(2):328-42. doi: 10.1111/j.1365-3040.2012.02576.x.

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