Anti-EIN3 | Ethylene insensitive 3

Samples:
MW markers: PageRuler™ from Thermo Scientific™
1. Arabidopsis thaliana wild-type Col-0
2. Arabidopsis thaliana ein2-1 mutant
3. Arabidopsis thaliana ein3-1 mutant
4. Arabidopsis thaliana eil1-1 mutant
5. Arabidopsis thaliana ein3-1/eil1-1 mutant
Total proteins were extracted freshly from 10-day-old Arabidopsis thaliana seedlings with plant protein extraction buffer (50 mM Tris/HCl pH7.5, 150mM NaCl, 1% Triton-X100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM DTT, 1% Thermo Scientific™ Halt™ Protease and Phosphatase Inhibitor Cocktail and 50 uM MG132). Protein concentrations of the samples were determined by Bio-Rad RC/DC™ Protein Assay kit and adjusted to the same concentration. The adjusted samples were 1:1 mixed with 2x SDS sampling buffer (125 mM Tris/HCl pH6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.2 mg/ml bromophenol blue) and denatured at 95 °C for 10 min. 40 ug of proteins per well were separated on Bio-Rad 4–20% Mini-PROTEAN® TGX™ gel and blotted 1h to PVDF (pore size of 0.45 µm) using wet tank transfer. Blot was blocked with 10% no-fat dry milk in TBS for 1h/RT with agitation. Blot was incubated in anti-EIN3 primary antibody (AS23 4896) at a dilution of 1: 1,000 in TBS + 3% BSA overnight at 4 °C with agitation. The antibody solution was decanted, and the blot was washed 3 times for 10 min each in 20 ml of TBS-T at RT with agitation. Blot was incubated with Invitrogen™ Goat anti-Rabbit IgG (H+L) HRP-conjugated secondary antibody diluted to 1:10,000 in TBS with 10% non-fat drymilk for 1h/RT with agitation. The blot was washed 4 times for each in 20 ml of TBS-T at RT with agitation and developed for 5 min with chemiluminescent detection reagent of high sensitivity. Exposure time was 15 seconds by using Bio-Rad ChemiDoc system.

Courtesy of Dr. Jian Yao, DOE-Plant Research Laboratory, Michigan State University, USA