Plant/Algal cell antibodies / Photosynthesis / PSI (Photosystem I)

product information

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.

This product is a recombinant protein standard, source: Synechocystis PCC 6803.

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store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.

western blot (WB)

collection of other protein standards

AS04 042 | anti-PsaC | PSI-C core subunit of photosystem I global rabbit antibodies

collection of other global antibodies

collection of antibodies to PSI proteins

Global antibodies are raised against highly conserved amino acid sequences in the PsaC protein. The PsaCprotein standard can therefore be used in combination with global anti-PsaC antibodies to quantitate PsaC from a wide range of species.

Quantitative western blot:  detailed method description.

application information

standard curve: 3 loads are recommended (0.5, 2 and 4μl).
For most applications a sample load of 0.2μg of chlorophyll will give a PsaC signal in this range.

positive control: a 2μl load per well is optimal for most chemiluminescent detection systems. 

This standard is stabilized and does not require heating before loading on the gel.

11.5 kDa (larger than native protein due to the addition of His-tag). In most gels PsaC migrates between 9 and 14 kDa

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no confirmed exceptions from predicted reactivity known in the moment

Concentration: after adding 225  µl of milliQ water final concentration of the standard is 0.09 pmoles/ul

Protein standard buffer composition: Glycerol 10%, Tris Base 141 mM, Tris HCl 106 mM, LDS 2%, EDTA 0.51 mM, SERVA® Blue G250 0.22 mM, Phenol Red 0.175 mM, pH 8.5, 0.1mg/ml PefaBloc protease inhibitor (Roche), 50mM DTT.

Bouchard et al. (2006) UVB effects on the photosystem II-D1 protein of phytoplankton and natural phytoplankton communities. Photochem and Photobiol 82: 936-951.  Morash et al. (2007) Macromolecular dynamics of the photosynthetic system over a seasonal developmental progression in Spartina alterniflora. Can J. of Bot. 85: 476-483(8)
 

application example

total protein from Trichodesmium sp. (1) and Thalassiosira sp.  (2). Recombinant PsaC protein standard (AS04 042S) (3-6) loaded at 0.5 pmoles, 0.3 0.1 and 0.05 pmoles. Molecular weight markers (MagicMark XP, Invitrogen) (7).  Samples 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: 50 000 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-hen IgY horse radish peroxidase conjugated, from Abcam) diluted to 1:50 000 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).
Note: Optimal quantitation is achieved using moderate sample loads per gel lane, generally 0.5 to 2.5 ug total protein, depending on the abundance of the target protein.