PsaC | PSI-C core subunit of photosystem I
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KLH-conjugated synthetic peptide conserved in all known PsaC proteins including Arabidopsis thaliana Uniprot:P62090 TAIR: AtCg01060, Hordeum vulgare UniProt: P69416, Oryza sativa UniProt: P0C360, Chlamydomonas reinhardtii UniProt: Q00914, Synechococcus elongatus UniProt: Q31QV2
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5 µg of total protein from samples such as (1) Arabidopsis thaliana leaf, (2) Hordeum vulgare leaf, (3) Chlamydomonas reinhardtii total cell, (4) Synechococcus sp. 7942 total cell, (5) PsaC protein standard (AS04 042S), total protein from all the samples were extracted with Agrisera Protein Extraction Buffer PEB (AS08 300). Samples were diluted with 1X sample buffer (NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM DTT and heat at 70°C for 5 min and kept on ice before loading. Protein samples were separated on NuPAGE 4-12% Tris-Bis gel (Invitrogen) LDS-PAGE and blotted for 1h to 1.5h on PVDF using tank transfer. Blots were blocked immediately following transfer in 2% blocking reagent for 1h at RT with agitation. Blots were incubated with PsaC antibody at a dilution of 1: 10 000 (in blocking reagent) for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed 1x15 min and 3x5 min with TBS-T at RT with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG HRP conjugated, AS09 602) diluted to 1:50 000 in blocking reagent for 1h at RT with agitation. The blots were washed as above. The blot was developed for 5 min with chemiluminescence detection reagent in mid picogram range. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).
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PsaC is a conserved, chloroplast-encoded, Fe-S binding protein of approximately 10kDa, present in all known Photosystem I complexes. It is located on the stromal side of the thylacoid membranes. PsaC coordinates the Fe–S clusters FA and FB through two cysteine-rich domains.
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