HSP21 | Chloroplastic heat shock protein

David Macherel

We have been using in our team this antibody several times on western blots to analyse the heat shock response in Arabidopsis. Leaf total protein extracts (Phosphaate 50 mM pH 8, 10 mM EDTA, 0.1 % Triton X100, 0.1 % Sarkosyl, 10 mM DTT, 1 mM PMSF and anti-protease cocktail) were prepared from plants subjected to a 3h heat (38°C) treatment followed by 2h of recovery. 20-40 µg proteins were separated by SDS-PAGE (13.5 %) and transferred onto PVDF membrane in CAPS buffer (100V, 1h). After red Ponceau staining, membranes were blocked for 1h with 1.5 % Tween 20 in TBS, rinsed with TBST (TBS with 0.05 % Tween). Incubation with HSP21 antibody (1/1000) was performed in TBST (2 h room temperature, or overnight 4°C). The secondary antibody (Agrisera goat anti-rabbit IgG HRP conjugate) was used at 1/75000 (1h, in TBST, room t°C). After TBST rinsing, revelation was performed using the Clarity western ECL kit (Biorad) and a Chemidoc (Biorad). A strong signal was rapidly detected at the expected size in the heat treated samples after a few min incubation. Although longer exposure could reveal some background (Rubisco?), the antibody is very useful to detect the heat induction of this plastidial small HSP.

2016-09-21 @ 12:27:34