Anti-SRK2E | Ser/Thr-protein kinase SnRK2,6

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 50 µM + ABA 50 µM, 6 hours)
3 - 50 µg of Arabidopsis thaliana ost1(snrk2.6) mock-treated (MG132 50 µM, 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 50 µM, 6 hours)
The ost1-3 (SALK_008068) and the abi1-2 (SALK_72009) mutants were used as controls.

50 µg/well of total protein extracted freshly from Arabidopsis thaliana roots with extraction buffer containing: 150 mM NaCl, 50 mM 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, Agrisera) diluted to 1: 10000 in for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: AgriseraECL SuperBright (AS16 ECL-S-10, Agrisera). Exposure time was 30 seconds.

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

Bacterial lysates were separated on 12% SDS-PAGE  and blotted 1h to PVDF using semi-dry or tank transfer. Blots were blocked with 5 % milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 3 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in PBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602 from Agrisera) diluted to 1:50 000 in  for 30 min. at RT with agitation. The blot was washed as above and developed for 3 min with ECL according to the manufacturer's instructions. Exposure time was 30 seconds.

Courtesy of Dr. Agnieszka Ludwików, UAM, Poznań, Poland

Protein A agarose beads (40µl) where coated with 10µl (1µg/ul) antibodies and after incubation with amount of extract (10 mg/ml) indicated washed extensively and loaded on gel. In gel kinase assay was performed as described in Fujii, 2007.
Autoradiograph shows immunoprecipitated kinase from plant extracts. 1 beads with BSA 20 µl loaded on gel 2 beads with plant extract (WT) 20 µl loaded on gel 3 beeds with plant extract (mutant X) 20 µl loaded on gel 4 beads with BSA 10 µl loaded on gel 5 beads with plant extract (WT) 10 µl loaded on gel 6 beeds with plant extract (mutant X) 10 µl loaded on gel 7 beads with BSA 5 µl loaded on gel 8 beads with plant extract (WT) 5 µl loaded on gel 9 beeds with plant extract (mutant X) 5 µl loaded on gel

Courtesy of Dr. Szymon Świeżewski, Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland