Journal: The Journal of biological chemistry

Article Title: The plastid-encoded ccsA gene is required for heme attachment to chloroplast c-type cytochromes.

doi: 10.1074/jbc.271.9.4632

Figure Lengend Snippet: FIG. 5. Phenotype of a strain (DccsA) containing a disrupted ccsA gene. Soluble proteins were prepared from copper-supplemented (1Cu) or copper-deficient (2Cu) cultures of the indicated strains and analyzed for the accumulation of holocytochrome c6 (heme stain and anti-cytochrome c6) and plastocyanin (anti-pc). Nitrocellulose membranes were used for the leftmost three panels. Bound antibody was detected by use of a horseradish peroxidase-conjugated secondary antibody. The pellet fractions from the preparation were analyzed for cytochrome f accumulation (anti-cytochrome f). PVDF membranes were used for the transfer, and an alkaline phosphatase-conjugated secondary antibody was used for detection.

Article Snippet: Bound antibodies were detected with alkaline phosphatase- or horseradish peroxidase-conjugated secondary antibody according to instructions provided by the manufacturer (Bio-Rad).

Techniques: Staining

Journal: The Journal of biological chemistry

Article Title: The plastid-encoded ccsA gene is required for heme attachment to chloroplast c-type cytochromes.

doi: 10.1074/jbc.271.9.4632

Figure Lengend Snippet: FIG. 6. Complementation of strain B6 with the cloned ycf5 gene. A, sum- mary of the complementation experi- ments. Plasmids pEBP and pEBH were constructed in vector pTZ19R, while pNH was constructed in the vector pET22b(1). The indicated frequencies are the aver- ages from three different experiments. B, extracts of soluble protein were prepared from copper-deficient cultures of strain CC425 (wt), strain B6, or cells of B6 res- cued by plasmid pEBP (B6-P) or pEBH (B6-H). Equivalent amounts (correspond- ing to 1 A595 unit in the Pierce Coomassie dye binding assay) were analyzed, after separation of proteins in an SDS-contain- ing polyacrylamide gel and transfer to a nitrocellulose membrane, for accumula- tion of holocytochrome c6 by heme stain- ing (bottom panel, 45-min exposure to NEN Reflection film) or decoration with anti-cytochrome c6 (top panel, horse- radish peroxidase conjugated second antibody).

Article Snippet: Bound antibodies were detected with alkaline phosphatase- or horseradish peroxidase-conjugated secondary antibody according to instructions provided by the manufacturer (Bio-Rad).

Techniques: Clone Assay, Construct, Plasmid Preparation, Binding Assay, Membrane, Staining