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Description
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Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes therate-limiting step of CO2 fixation in photosynthesis. It is one of the most abundantproteins on Earth and its homology has been demonstrated from purple bacteriato flowering plants. Source of a standard: RbcL protein purified from spinach. immunogen antibody format quantity 250 µl lyophilized, for reconstitution add 225 µl of milliQ water storage store lyophilized/reconstituted at -20°C, once reconstituted make aliquots to avoidrepeated freeze-thaw cycles. Please, remember to spin tubes briefly prior toopening them to avoid any losses that might occur from lyophilized materialadhering to the cap or sides of the tubes. tested applications western blot (WB) additional information Global antibodies are raised against highly conserved amino acid sequences inthe RbcL protein. The RbcL protein standard can therefore be used incombination with global anti-RbcL antibodies to quantitate RbcL from a widerange of species. Quantitative western blot: detailed method description. application information recommended dilution 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 signalin this range. positive control: a 2µl load per well is optimal for most chemiluminescent detectionsystems. This standard is stabilized and does not require heating before loading on the gel. expected | apparent MW 52.7 kDa confirmed reactivity predicted reactivity not reactive in no confirmed exceptions from predicted reactivity currently known 09/22/10 21:45:10 additional information Concentration: after adding 225 µl of milliQ water final concentration of thestandard is 0.15 pmoles/µl Protein standard buffer composition: Glycerol 10%, Tris Base 141 mM, Tris HCl106 mM, LDS 2%, EDTA 0.51 mM, SERVA® Blue G250 0.22 mM, Phenol Red0.175 mM, pH 8.5, 0.1mg/ml PefaBloc protease inhibitor (Roche), 50mM DTT. selected references MacKenzie et al (2005). Large reallocations of carbon, nitrogen andphotosynthetic reductant among phycobilisomes, photosystems and Rubiscoduring light acclimation in Synechococcus elongatus are constrained in cellsunder low environmental inorganic carbon. Arch of Microbiol. 183: 190 - 202.Bouchard et al. (2006) UVB effects on the photosystem II-D1 protein ofphytoplankton and natural phytoplankton communities. Photochem and Photobiol82: 936-951. Morash et al. (2007) Macromolecular dynamics of thephotosynthetic system over a seasonal developmental progression in Spartinaalterniflora. 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. MarkersMagicMarks (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 CCDimager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad). The contour tool ofthe software is used to select the area for quantitation and the values are backgroundsubtracted 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. Quantitation: When quantitated standards are included on the blot, the samples can be quantitated using theavailable software. Excellent quantitation can be obtained with images captured on the Bio-Rad Fluor-S-Max orequivalent instrument using Bio-Rad QuantityOne software. The contour tool is used to select the area forquantitation and the values are subtracted to give an adjusted volume in counts for each standardand sample. Using above protocol linear standard curves are generated over 1-1.5 orders of magnitude rangein target load. It is important to note that immunodetections usually show a strongly sigmoidal signal to loadresponse curve, with a region of trace detection of low loads, a pseudolinear range and a region of saturatedresponse with high loads. For immunoquantitation it is critical that the target proteins in the samples and thestandard curve fall within the pseudolinear range. Our total detection range using this protocol spans over 2orders of magnitude, but the quantifiable range is narrower. Quantitative western blot: detailed method description. 09/22/10 21:45:10
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