Heat Stress Protein Analysis in Arabidopsis

Arabidopsis leaves were collected before and after heat treatment, ground in liquid nitrogen and homogenized in an detergemt-containing extraction buffer (100 mMTris/HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.2% ß-mercaptoethanol). The homogenates were filtered through a 300 µm and 100 µm nylon mesh and clarified by centrifugation at 2,200xg for 5 minutes. Supernatants were kept for further analysis. The pellets were resuspended in the same buffer and subjected to the low speed centrifugation. The process was repeated twice and after the last centrifugation the pellets were resuspended in the extraction buffer. The concentrations of proteins in the homogenates (total proteins), the first supernatants (soluble proteins) and last pellets (insoluble proteins) were determined using Bio-Rad protein assay kit. The first supernatant fractions and last pellets were separated by SDS–PAGE. For western blotting, fractionated proteins on SDS/PAGE gel were transferred to nitrocellulose membrane. NBR1-TAP was detected by a peroxidase-conjugated anti-peroxidase antibody. Ubiquitinated proteins were detected by protein blotting using an anti-ubiquitin monoclonal antibody (Sigma, USA). The antigen-antibody complexes were detected by enhanced chemiluminescence using luminal as substrate as previously described [39] .