2 | Rubisco quantitation kit
AS09 409 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Dicots, monocots, algae, cyanobacteria
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1 x 100 µl of AS01 017S, Rubisco protein standard (0.15 pmoles / µl, amount enough for generation of standard curve in 34 assays (standard curve: 0.0625 pmoles, 0.125 pmoles, 0.25 pmoles)
2 x 2 ml of AS08 300, Protein extraction buffer (amount enough for 48 isolations of plant material, using 500 µl 1x PEB for 100 mg fresh weight) or 120 isolations of algal material (using 200 µl 1x PEB for cell amounts corresponding to 4-10 µg total chlorophyll)
1 x 50 µl of AS09 602, Goat anti-rabbit IgG (H&L), HRP conjugated(amount enough for 50-100 Western blots)
1 X 10 ml of AS16 ECL-N-10, AgriseraECL Bright (10 ml trial pack)
Rubisco protein standard: 52.7 kDa (Arabidopsis thaliana), 52.5 kDa (cyanobacteria), 52.3 (Chlamydomonas reinhardtii)
2 µg of total protein from various plant extracts (1-5) extracted with Agriera Protein Extraction Buffer 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.
The Rubisco quantitation kit is suitable for quantitation of Rubisco in plant and algal samples using quantitative immunoblotting. It contains anti-RbcL antibody, Rubisco protein standard and the Protein Extraction Buffer for quantitative protein extraction. This kit is a good alternative to quantification of Rubisco by ELISA as it allows separation of Rubisco from a total complex cell protein mixture, which can be troublesome in case of ELISA method.
Mota et al. (2015). Effects of heavy metals on Cyanothece sp. CCY 0110 growth, extracellular polymeric substances (EPS) production, ultrastructure and protein profiles. J Proteomics. 2015 Apr 29;120:75-94. doi: 10.1016/j.jprot.2015.03.004.
Thamatrakoln et al. (2013). Death-specific protein in a marine diatom regulates photosynthetic responses to iron and light availability. Proc Natl Acad Sci U S A. 2013 Dec 10;110(50):20123-8. doi: 10.1073/pnas.1304727110. Supplemental material describes western blot quantification method.