DS5a | Drosophila 26S proteasome subunit Rpn10

Drosophila mealnogaster 26S Proteasome subunit S5a also known as Rpn10, p54. Antigen sequence is also conserved in Arabidopsis thaliana AT4G38630

42.6 | 54 kDa (Drosophila), 40 kDa (A. thaliana)

Application example

3 µg of total protein from  Arabidopis thaliana leaves  extracted with CelLytic P (Sigma)  were separated on 10  % SDS-PAGE and blotted overnight at 30V to a nitrocellulose membrane. Blots were blocked with 1% western blocking reagent (Roche) for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody (DS5a) at a dilution of 1:2000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly, and then washed twice for 10 min with TBS-T at RT with agitation. Subsequently, the blot was washed twice with 0,5% western blocking reagent for 10 min each. Blot was incubated in secondary antibody (Goat anti-rabbit IgG (H&L) HRP conjugated, AS09 602 from Agrisera) diluted to 1:50 000 in 0,5% western blocking reagent for 1h at RT with agitation. The blot was washed 4 times with TBS-T buffer. Detection of HRP was performed with ECL according to the manufacturer's instructions. Exposure time was 2 seconds. Position of protein on gel corresponds to expected size - 40 kDa.

Courtesy Dr. Jozefus Schippers, Max Planck Institute, Germany

Proteasome-dependent degradation serves an essential role in the removal of a wide variety of key nuclear and cytosolic proteins. Substrates are targeted for proteolysis by the ubiquitin pathway before being degraded by the 26 S proteasome. The subunit of the proteasome, S5a, was identified to bind to polyubiquitin in vitro and thus proposed to act as a substrate recognition component.