HSP23.5 | Heat shock protein 23.5 (mitochondrial)

Samples: 

1- 20 μg of Arabidopsis thaliana Col-0 whole protein extract
2- 20 μg of an Arabidopsis thaliana Hsp23.5 null mutant whole protein extract
3- 20 μg of an Arabidopsis thaliana Hsp23.6 null mutant whole protein extract
4- 20 μg of an Arabidopsis thaliana Hsp23.5 and HSp23.6 double null mutant whole protein extract
5- 20 μg of an Arabidopsis thaliana Hsp23.5, 23.6 and 26.5 triple null mutant whole protein extract

Protein for lanes 1-5 was extracted from plants kept at RT

6- 20 μg of Arabidopsis thaliana Col-0 whole protein extract
7- 20 μg of an Arabidopsis thaliana Hsp23.5 null mutant whole protein extract
8- 20 μg of an Arabidopsis thaliana Hsp23.6 null mutant whole protein extract
9 - 20 μg of an Arabidopsis thaliana Hsp23.5 and HSp23.6 double null mutant whole protein extract
10 - 20 μg of an Arabidopsis Hsp23.5, 23.6 and 26.5 triple null mutant whole protein extract

Protein for lanes 6-10 was extracted from plants heat stressed at 38°C for 1 hour, left to recover at RT for 3 hours and then flash frozen.

20 μg/well of total protein extracted from 10 day old Arabidopsis thaliana seedlings (WT and mutant, flash frozen in liquid nitrogen and stored at -85°C) with 60 mM Tris-HCl pH 6.8, 60 mM DTT, 2% SDS, 1.5% Sucrose and 0.05% bromophenol blue and denatured with 1X SDS Loading Dye (10% glycerol, 0.002% bromophenol blue, 0.5% SDS, 50 mM Tris-HCl pH 6.8) at 95°C for 5 min were separated on 4- 20% SDS-PAGE (nUView Tris-Glycine Precast Gel, NuSep) and blotted 1h to nitrocellulose (0.45 μm pore size) using semi-dry transfer. Blot was blocked with 5% milk in TBS-T for 15 minutes, blocking solution was replaced and blocked for another 15 minutes for 30 minutes total with agitation. The blot was incubated in the primary antibody at a dilution of 1:1000 for ~6 hours at 4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed for 10 minutes, a process repeated three times in total for 30 minutes of washes with TBS-T at RT with agitation. Blot was incubated in Goat Anti- rabbit IgG (horse radish peroxidase conjugated) diluted 1:5000 in TBS-T with agitation. The blot was washed as above and developed for 2 minutes in homemade ECL solution (1.25 mM Luminol, 0.1 M Tris pH 8.5, 0.2 mM p-coumaric acid, 1 mL of solution mixed with 3 μL of 3% hydrogen peroxide just before development). Exposure time was 11 minutes in a G:Box iChemi XT (Syngene).

Courtesy of Fabian Suri-Payer, Vierling Lab,Department of Biochemistry & Molecular Biology University of Massachusetts Amherst, USA