Anti-HSP101 | ClpB heat shock protein, N-terminal (chicken antibody)

Samples: 

(1) Plants grownat 18°C
(2) Same plantsafter 1.5 h at 38°C
(3) Same plants after 2 h at 22°C
(4) Same plantsafter3 h at 45°C
(5) Sameplants after 4 days of recovery

40 μg protein of total leaf extracts of Arabidopsis thaliana was separated on a 13.5% Laemmli SDS-PAGE gel and transferred to nitrocellulose (0,2 μM pore size). The membrane was blocke dwith 5% dry-milk in 1xPBS for 1h at room temperature (RT). The membrane was incubated with primary antibody(Agrisera AS08 283) diluted 1: 5 000 in 5% dry-milk in 1xPBS for 1h at RT. After washing with 1xPBS the membrane was incubated with secondary antibody (Agrisera AS09 603) Goat anti-chicken IgY (H&L), HRP conjugated diluted1:10 000in 1x PBS with 1% dry-milk for 1 h at RT. After washing with 1xPBS the blot was developed with chemiluminescent detection reagent. Exposure time: 60 seconds. 

2 µg of total protein from (1) Arabidopsis thaliana WT stressed at 22ºC, (2) Arabidopsis thaliana WT stressed at 38ºC for 1.5 hour followed by a transfer to 22° for 2 hours, (3) Arabidopsis thaliana Hsp101 null mutnat grown at 22°C, (4) Arabidopsis thaliana Hsp101 null mutant stressed at 38ºC for 1.5 hour followed by a transfer to 22ºC for 2 hours were separated on 8 % SDS-PAGE and blotted 1h to nitrocellulose (Biorad). Blots were incubated in the primary antibody at a dilution of 1: 1 000 for 1h at room temperature with agitation and a secondary HRP-conjugated antibody (1: 10 000).

Hsp101/ClpB is a member of HSP100 protein family. These proteins help protein aggregates formed during heat stress to fall apart to allow them to be refolded by other chaperones. In spite of expression during heat stress members of HSP100 protein family are also expressed during seed development.