Plant/Algal cell antibodies / Photosynthesis / RUBISCO

product information

Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. It is one of the most abundant proteins on Earth and its homology has been demonstrated from purple bacteria to flowering plants.

Source of a standard: RbcL protein purified from spinach.

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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

AS01 017 | anti-RbcL Type I hen global antibody

AS03 037 | anti-RbcL Type I and II rabbit global antibody

collection of other global antibodies

collection of antibodies to photosythetic proteins

Global antibodies are raised against highly conserved amino acid sequences in the RbcL protein. The RbcL protein standard can therefore be used in combination with global anti-RbcL antibodies to quantitate RbcL 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 RbcL signal in this range.

positive control: a 2μl load per well is optimal for most chemiluminescent detection systems. Higher standard concentration needs to be used to allow detection by Coomasie stains. Such gels with higher standard concentration can not be used for quantitation using chemiluminescence.

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

52.7 kDa

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no confirmed exceptions from predicted reactivity currently known

Concentration: after adding 225  µl of milliQ water final concentration of the standard is 0.15 pmoles/µl

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.

Rubisco protein was purified directly from plant tissue.

MacKenzie et al  (2005). Large reallocations of carbon, nitrogen and photosynthetic reductant among phycobilisomes, photosystems and Rubisco during light acclimation in Synechococcus elongatus are constrained in cells under low environmental inorganic carbon. Arch of Microbiol. 183: 190 - 202. 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

2 µg of total protein from various plant extracts  (1-5) extracted with PEB (AS08 300)  separated on  4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Markers MagicMarks (Invitrogen) (M) and  Rubisco protein standard (AS01 017S) at 0.0625 pmol, 0.125 pmol, 0.25 pmol.

Following standard western blot procedure this image has been obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).  The contour tool of the software is used to select the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample.

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.