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Fig. 2. In vitro binding analyses of SBP1 and SKD1 (A) Two hybrid binding analyses. Rectangles represent the protein structure of SBP1 (309 amino acids) and the five clones that were obtained from yeast two-hybrid screening. Mav203 cells transformed with pDBLeu-SKD1 and pPC86-SBP1 (clone 1-5) were plated on SC His+ Trp- Leu- (1, 2), SC His- Trp- Leu- (3) and SC His- Trp- Leu- Ura- (4) plates. An X-gal assay was performed on the nitrocellulose filter (2). Mav203 cells, transformed with the two plasmids supplied with the kit (pPC97-Fos and pPC86-Jun) as a positive control (P.C.), or with the empty vector pPC86 and pDBLeu-SKD1 as a negative control (pPC86) are indicated. (B) SPR spectroscopy. Indicated concentrations (30, 60, and 150 µg/ml) of His-thioredoxin-SBP1 (full length) in binding buffer were injected over the immobilized GST-SKD1 on a CM5 sensor chip. An arrowhead and arrow indicate the sample and dissociation buffer injection points, respectively. (C) GST pull-down assay. GST or GST-SKD1 was immobilized on glutathione-sepharose prior to incubation with purified His-thioredoxin-SBP1s [full length (1-309), 
N (198-309), and C (1-129)]. Bound proteins were eluted with SDS sample buffer and processed for immunoblotting with anti-SBP1 antibody.
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