RbcL | Rubisco positive control/quantitation standard
AS01 017S | protein standard for quantitation and positive control
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Standard curve: 3 loads are recommended (0.5, 2 and 4μl).
Positive control: a 2 μl 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 does not require heating before loading on the gel or addition of any buffer.
|Expected | apparent MW||
|Not reactive in|
Concentration: after re-constitution with sterile 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), 50 mM DTT.
Please, use the 55 kDa size of RbcL for calculations. The pmoles in the standard refer to pmoles of rbcL monomers.
|Selected references||Li et al. (2016). Interactive effects of nitrogen and light on growth rates and RUBISCO content of small and large centric diatoms. Photosynth Res. 2016 Aug 26. [Epub ahead of print]
Young et al. (2015). Antarctic phytoplankton down-regulate their carbon-concentrating mechanisms under high CO2 with no change in growth rates. Marine Ecology Progress Series 532:13-28.
Vandenhecke et al. (2015). Changes in the Rubisco to photosystem ratio dominates photoacclimation across phytoplankton taxa. Photosynth Res. 2015 Apr 11.
Wu et al. (2014). Large centric diatoms allocate more cellular nitrogen to photosynthesis to counter slower RUBISCO turnover rates. Front. Mar. Sci., 09 December 2014 | doi: 10.3389/fmars.2014.00068.
Li et al. (2014). The nitrogen costs of photosynthesis in a diatom under current and future pCO2. New Phytol. 2014 Sep 25. doi: 10.1111/nph.13037.
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 the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample.
Quantitation: When quantitated standards are included on the blot, the samples can be quantitated using the available software. Excellent quantitation can be obtained with images captured on the Bio-Rad Fluor-S-Max or equivalent instrument using Bio-Rad QuantityOne software. The contour tool is used to the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample. Using above protocol linear standard curves are generated over 1-1.5 orders of magnitude range in target load. It is important to note that immunodetections usually show a strongly sigmoidal signal to load response curve, with a region of trace detection of low loads, a pseudolinear range and a region of saturated response with high loads. For immunoquantitation it is critical that the target proteins in the samples and the standard curve fall within the pseudolinear range. Our total detection range using this protocol spans over 2 orders of magnitude, but the quantifiable range is narrower.
Quantitative western blot: detailed method description.
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