AS12 1861 Anti-ABI1 | Abscisic acid insensitive 1

Confirmed reactivity:	Arabidopsis thaliana
Not reactive in:	Sorghum bicolor

Samples: 1 - 50 µg of Arabidopsis thaliana Col0 mock-treated (MG132 50 µM, 6 hours)
2 - 50 µg of Arabidopsis thaliana Col0 ABA-treated (MG132 50µM + ABA 50 µM, 6 hours)
3 - 50 µg of Arabidopsis thaliana ost1(snrk2.6) mock-treated (MG132 5 0µM, 6 hours)
4 - 50 µg of Arabidopsis thaliana ost1(snrk2.6) ABA-treated (MG132 50 µM + ABA 50µM 6, hours)
5 - 50 µg of Arabidopsis thaliana abi1-2 mock-treated (MG132 50 µM, 6 hours)
6 - 50 µg of Arabidopsis thaliana abi1-2 ABA-treated (MG132 50 µM + ABA 50µM, 6 hours)

50 µg/well of total protein extracted freshly from Arabidopsis thaliana roots with: 150 mM NaCl, 50mM Tris-HCL pH 8, 1% Triton X-100, anti-proteases cocktail (Complete mini EDTA free, “ROCHE”) (1 tablet for 10ml), 3 mM DTT, 50 mM MG132, or 50 mM ABA; and denatured with exact buffer components at 95°C/5 min.  Samples were separated on 10% SDS-PAGE and blotted overnight (ON) to PVDF (Inmobilon®-FL) (pore size of 0.45 µm), using: wet transfer. Blot was blocked with 3% milk for: 6h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10 000 in TBS-T 1X for ON/4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (Goat anti-rabbit IgG HRP conjugated, AS09 602, Agroisera) diluted to 1: 10 000 in for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: Agrisera ECL SuperBright (AS16 ECL-S-10) supplied by Agrisera. Exposure time was 10 seconds.

Courtesy of Drs. Javier Ocaña, Alberto Coego and Pedro L. Rodriguez, CSIC, Spain

Additional information:	It is of crucial importance to chose a material in which ABI1 protein is highly expressed like seeds or senescent leaf. This protein could not be detected using this antibody in plants grown under optimal (non stressed) conditions, The antibody detects both, recombinant and endogenous ABI1 proteins.
Additional information (application):	Important note: blocking with more than 3% skimmed milk will result in lack of signal for this antibody

ABI1 is a key component and repressor of the abscisic acid (ABA) signaling pathway. It regulates numerous ABA responses, such as stomatal closure, osmotic water permeability of the plasma membrane, drought-induced resistance and rhizogenesis, response to glucose, high light stress, seed germination and inhibition of vegetative growth. Expressed in seeds and seedlings. Confined to lateral root caps and columella cells in roots. Induced by low temperature, drought, high salt, ABA and ethylene.  Activates/represses SnRK2.6/SRK2E/OST1 in response to ABA-dependent stimuli. Alternative names: ABI1, ABA INSENSITIVE 1, AtABI1, Protein phosphatase 2C 56, AtPP2C56, PP2C56.

1. Mitula et al. (2015). Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin-Proteasome Pathway. Plant Cell Physiol. 2015 Dec;56(12):2351-67. doi: 10.1093/pcp/pcv146. Epub 2015 Oct 6.