CP43' | IsiA homolog of plant CP43
AS06 111 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Cyanobacteria
|Recommended dilution||1 : 500-1 : 1000 (WB)|
|Expected | apparent MW||
37 | 27 kDa (in a Novex gel system)
|Confirmed reactivity||Acaryochloris marina, Chlamydomonas reinhardtii, Synechococcus elongatus PCC 7942, Synechocystis sp. PCC 6803|
|Not reactive in||No confirmed exceptions from predicted reactivity are currently known.|
|Selected references||Xing et al. (2017). Deletion of CGLD1 Impairs PSII and Increases Singlet Oxygen Tolerance of Green Alga Chlamydomonas reinhardtii. Front. Plant Sci., 15 December 2017.
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.
5 µg of total protein from (1) Synechocystis sp. PCC 6803 extracted with PEB (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% ECL Advance blocking reagent (GE Healthcare) 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% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance detection reagent 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 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 (GE RPN 2125; Healthcare) 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 TMA-6 (Lumigen) detection reagent 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.
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