CP43' | IsiA homolog of plant CP43
AS06 111 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Cyanobacteria
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KLH-conjugated synthetic peptide nearly perfectly conserved across known IsiA/CP43 proteins including Synechocystis PCC sp. 6803 CP43' Q55274
37 | 27 kDa (in a Novex gel system)
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5 µg of total protein from (1) Synechocystis sp. PCC 6803 extracted with Agrisera Protein Extration Buffer (AS08 300), (2) Synechocystis sp. PCC 6803 grown in low iron culture, extracted with PEB), were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% blocking reagent in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 1000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:20 000 in 2% blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with chemiluminescence detection reagent of extreme femtogram sensitivity, according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).
Cross-reacting band above 50 kDa is Rubisco.
Total protein (2.5 µg) from Synechocystis PCC 6803 was 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 before loading. Protein samples were separated on 4-12% Bolt Plus gels, LDS-PAGE and blotted for 60 minutes to PVDF using tank transfer. Blots were air-dried until further processing, then rehydrated with methanol and blocked in 2% blocking reagent dissolved in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 2500 (in blocking reagent) for 1h at room temperature 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 room temperature with agitation. Blots were incubated in secondary antibody goat anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS09 602, Agrisera) diluted to 1:7500 in blocking reagent for 1h at room temperature with agitation. The blots were washed as above. The blot was developed with chemiluminescent detection reagent of extreme femtogram sensitivity, according the manufacturer’s instructions. Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 60 seconds.
Peptide used to elicit this antibody is also perfectly or highly conserved in known Pcb chlorophyll a/b binding proteins from Prochlorococcus and similar proteins from other cyanobacteria. Peptide target is partially conserved in CP43/PsbC. CP43' and CP43 can be distinguished by their size.
CP43' is encoded by the IsiA gene. This chlorophyll (Chl)-binding protein is induced under iron deficiency conditions in certain cyanobacterial strains. It acts as dissipater of light energy protecting photosystem II from excessive excitation under iron-deficient conditions.
Li et al. (2017). The identification of IsiA proteins binding chlorophyll d in the cyanobacterium Acaryochloris marina. Photosynth Res. 2017 Apr 4. doi: 10.1007/s11120-017-0379-6.
Cheng and He (2014). PfsR Is a Key Regulator of Iron Homeostasis in Synechocystis PCC 6803. PLoS One. 2014 Jul 10;9(7):e101743. doi: 10.1371/journal.pone.0101743. eCollection 2014.
Selão et al. (2014). Subcellular Localization of Monoglucosyldiacylglycerol Synthase in Synechocystis sp. PCC6803 and Its Unique Regulation by Lipid Environment. PLoS One. 2014 Feb 6;9(2):e88153. doi: 10.1371/journal.pone.0088153. eCollection 2014.
Hakkila et al. (2013). Group 2 sigma factor mutant ΔsigCDE of the cyanobacterium Synechocystis sp. PCC 6803 reveals functionality of both carotenoids and flavodiiron proteins in photoprotection of photosystem II. Plant Cell Physiol. Sept 4.
Fraser et al. (2013). Photophysiological and Photosynthetic Complex Changes during Iron Starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942. PLoS One.
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