Anti-DnaK 抗体 (ab80161)

Lane 1. 15 µg of Arabidopsis thaliana leaf extract.

Lane 2. 10 µg of total protein from: Synechocystis 6803 motile.

Lane 3. Synechocystis 6803 GT (glucose tolerant strain).

Lane 4. Synechococcus elongates 7942.

Marker – Pierce™ Pre-stained Protein MW Marker (kat #26612)

Total protein was extracted with following buffer: 10 mM Tris HCl, pH 8.0, 0.5% LDS, 4% glycerol, 0.1 mM EDTA were mixed with sample buffer and denatured for 5 min at 95°C. Samples were separated on 10% SDS -PAGE and blotted for 1h to nitrocellulose membrane (Amersham Protran) using tank wet transfer (Bio-Rad) in standard transfer buffer in the presence of 20% methanol. Transfer of proteins to the membrane was checked using 0.5% Ponceau S staining before the blocking step. Blots were blocked in buffer (2% low-fat milk in 1xPBS, 0.1% Tween) for 1 h at room temperature (RT) with agitation. Blots were incubated with the primary antibody (ab80161) at a dilution of 1/5000 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. The blot was incubated in secondary antibody goat anti-rabbit IgG, diluted to 1/30000 for 1h at RT with agitation. The blot was washed as above and developed for 5 min with Clarity Western ECL Substrate and ChemiDoc detection system (Bio-Rad).