HSP23.5 | Heat shock protein 23.5 (mitochondrial)
AS15 2979 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana
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HSP23.5 protein was synthesized in vitro using using the PURExpress In Vitro Protein Synthesis Kit (NEB) according to manufacturer instructions. 20 µL of reaction mixture were separated on 13.5 % SDS-PAGE, and blotted 1h to PVDF (0.2 µm Immobilon PSQ transfer membrane, Millipore) using tank transfer (10 mM CAPS pH 11, 10 % ethanol). Blot was blocked with TBS containing 1.5 % Tween 20 by incubation at room temperature (RT). Blot was briefly rinsed three times with TBS-T (TBS with 0.05 % Tween 20) and incubated overnight at 4°C with the primary antibody at a dilution of 1:1 000 in TBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 15 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (goat anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602) diluted to 1:75 000 in TBS-T for 1h at RT with agitation. The blot was washed as above and developed for 2 min with chemiluminescent detection reagent.
Note: Protein was synthesized in vitro using the PURExpress In Vitro Protein Synthesis Kit (NEB)
Courtesty Dr. David Macherel, Mitostress team, IRHS, France.
High sensitivity reagents and enrichment are necessary due to low amounts of HSP23.5 in endogenous tissue.
Antibody is not cross reacting with cytosolic HSPs.
Please, note that there might be no HSPs accumulation below temperature of 32-34°C. HSPs are induced when the plant experience temperatures higher than the growing temperature with around 10°C. So, the HSPs induction temperatures for plants grown at 18C differ from these for plants grown at 24C.
Another very effective parameter is the humidity. When using low humidity the plant has a chance to cool down through transpiration. In this case the HSPs induction requires higher temperatures.
HSP23.5 | heat shock protein 23.5 (mitochondrial) belongs to a small heat shock protein family HSP20.