Journal: PLoS ONE
Article Title: The Deinococcus radiodurans DR1245 Protein, a DdrB Partner Homologous to YbjN Proteins and Reminiscent of Type III Secretion System Chaperones
doi: 10.1371/journal.pone.0056558
Figure Lengend Snippet: A. SDS-PAGE analysis of protein complexes purified from GY12830 strain expressing a tagged DdrB-SPA protein. Purification of protein complexes containing DdrB-SPA was carried out from irradiated cells (3,800 Gy) at 45 minutes after irradiation. * This band corresponds to the tagged DdrB with calmodulin binding protein. B. Purified native DdrB (non-tagged) (10 nmol) linked to streptavidin agarose beads using biotinylated anti-DdrB IgY were incubated with 10 nmol purified native (non-tagged) D. radiodurans DR1245 protein. Following extensive washes with PBS-Tween, proteins that remained associated to DdrB were eluted using 10x PBS. Proteins contained in the flow-through (1) and elution (2) fractions were analyzed on a 12% SDS-PAGE revealed using Coomassie blue staining. Positions of DdrB, DR1245 and the small subunit of the IgY are indicated.
Article Snippet: Streptavidin agarose beads (Thermo) were saturated with biotinylated anti-DdrB affinity purified chicken polyclonal IgY antibody (GeneTel Laboratories, LLC, Madison) in 1x PBS-Tween (10 mM sodium phosphate, 150 mM NaCl, 0.05% Tween-20, pH 7.5) and incubated for 1 hour with agitation.
Techniques: SDS Page, Purification, Expressing, Protein Purification, Irradiation, Binding Assay, Incubation, Staining