MPK6 | Mitogen-activated protein kinase 6

Application example

0.2g of 21 days old Arabidopsis thaliana leaf tssue C=Col, 6= mpk6 (T-DNA mutant), 3= mpk3 (T-DNA mutant) was homogenized with 250 μ l Buffer A( 50 mM Tris – HCl pH 7.5, 0.33 M sucrose, 5 mM EDTA, 150 mM NaCl , complete protease inhibitor cocktail). The crude extracts were subjected to centrifugation at 10 000 g for 10 min to pellet the insoluble material.   Material was extracted with  250 μl of Buffer A (50 mM Tris – HCl pH 7.5, 0.33 M sucrose, 5 mM EDTA, 150 mM NaCl, complete protease inhibitor cocktail). The crude extracts were subjected to centrifugation at 10 000 g for 10 min to pellet the insoluble material. Supernatant (120 μ L)+ 4XSDS sample buffer 40 μ L= 200 μ L samples ↓ 95 °C 5min 10 μ L were separated on 10 % SDS-PAGE  and blotted 1h to PVDF using semi-dry transfer. Blots were blocked with skim milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody in TBS-T at a dilution of 1: 10 000 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 TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602) diluted to 1:75 000 in  for 1h at RT with agitation. The blot was washed as above and developed with chemiluminescence according to the manufacturer's instructions.

Courtesy of Chika Tateda, University of Chicago, USA