Anti-PDHE1-B | Pyruvate dehydrogenase E1 component subunit beta-1, mitochondrial

40 µg/well of total protein extracted freshly from Arabidopsis thaliana whole rosette leaves.   Exact buffer components were: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40 (NP-40), 1% (w/v) deoxycholate, 0.1% (w/v) SDS, 1 × Complete protease inhibitor cocktail (Roche), 1 mM PMSF, and denatured with 5xSDS sample buffer (300 mM Tris-HCL(pH 6.8), 10% SDS, 0.1% Bromophenol, 50% Glycerol, 500 mM DTT) at 95°C/5 min.  Samples were separated in the RT on 10 % SDS-PAGE, and blotted for 7 min to nitrocellulose (pore size of  0.2 um), using:  iBlotTM Dry Blotting System (Invitrogen) in the RT. Blot was blocked with 5% milk for: 3h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:  500 for ON/4°C with agitation in 2% milk. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed 5 times for 5 min in PBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 5000 in 2% milk for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: AS16 ECL-N-10 Agrisera Bright. Exposure time was 1 minute.

To increase signal/noise ratio, primary antibody can be used in a dilution of 1: 1000 or 1: 2000 1h/RT incubation.

Courtesy of Dr. Mengping Li, Department of Botany and Plant Biology, UNIGE, Switzerland