Anti-MKK1 | Mitogen-activated protein kinase kinase 1

Samples: 
Col - Arabidopsis thaliana wild type
OE-GFP-MKK1 - Arabidopsis thaliana overexpressing MKK1 tagged with GFP

20 μg of total protein extracted freshly from 7 days-old Arabidopsis thaliana seedlings with Lysis buffer (25 mM Tris-HCl pH 7.5, NaCl 150 mM, 1% Nonidet, 0.1% SDS and Protease inhibitor cocktails) and denatured with Laemmli buffer at 75ºC for 10 min. The samples were separated on 10% SDS-PAGE and blotted 1h to nitrocellulose (pore size: 0.45 μm) using wet transfer. Blot was blocked with 5% milk for RT/ 1h with agitation. Blot was blocked in the primary antibody at a dilution of 1: 500 for 4ºC/ON with agitation in PBS-T. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 10 min and 5 times for 10 min in PBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1: 25 000 for 1h/RT with agitation. The blot was washed as above and developed for 5 min with Agrisera ECLBright. Exposure time was 30 seconds.

Conditions for MKK1 detection in wild type samples need to be determined. Suggested is: increased protein load/well to 50 μg combined with primary antibody incubation 4ºC/ON and detection reagent of highest sensitivity. 

Alternative names: AtMKK1 Short name: MAP kinase kinase 1 Alternative name(s): AtMEK1 NMAPKK