2 | Rubisco quantitation kit

AS09 409 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Dicots, monocots, algae, cyanobacteria

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product information
Background		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.
Immunogen		KLH-conjugated synthetic peptide was used to elicit anti-RbcL antibody. No baculovirus was used in production of this product.
Host		Rabbit. Secondary antibody: Goat.
Clonality		Polyclonal
Clone
Purity
Format
Quantity		1 x 50 µl of AS03 037, RbcL \| Rubisco large subunit, form I and form II (amount enough for 50-100 Western blots) 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)
Reconstitution
Storage		RbcL antibody and protein standard: Please store at -20°C (6 month) or -80°C for long term storage(years). Please, avoid freezing and thawing of reconstituted antibodies, make aliquots instead.PEB extraction buffer:Stable at RT for at least 1 month. For short-term storage please stoore (1 month) at 4°C and for long term storage (1 year) at -20°C.
Tested applications		Western blot (WB)
Related products		AS03 037 \| anti-RbcL \| Rubisco large subunit, form I and form II (50 µl) AS03 037A \| anti-RbcL \| Rubisco large subunit, form I and form II (50 µg affinity purified) AS03 037-HRP\| anti-RbcL \| Rubisco large subunit, form I and form II (40 µg, HRP-conjugated) AS15 2994 \| Rubisco ELISA quantitation kit AS15 2955 \| anti-RbcL II \| Rubisco large subunit, form II (50 µl), rabbit antibody AS15 2955S \| RbcL II \| Rubisco form II positive control/quantitation standard AS01 017 \| anti-RbcL \| Rubisco large subunit, form I, chicken antibody AS01 017S \| Rubisco protein standard for quantitative western blot or positive control AS03 037PRE \| Rubisco large subunit, pre-immune serum AS07 218 \| anti-Rubisco \| 557 kDa hexadecamer, rabbit antibody to a whole protein AS07 259 \| anti-RbcS \| Rubisco small subunit (SSU), rabbit antibody AS07 222 \| anti-RbcS \| Rubisco small subunit (SSU) from pea, rabbit antibody matching secondary antibody
Additional information		Recommended secondary antibody: AS09 602, Goat anti-rabbit IgG (H&L), HRP conjugated

application information
Recommended dilution		1 : 5000-1 : 10000 (WB)
Expected \| apparent MW		Rubisco protein standard: 52.7 kDa (Arabidopsis thaliana), 52.5 kDa (cyanobacteria), 52.3 (Chlamydomonas reinhardtii)
Confirmed reactivity		Arabidopsis thaliana, Chlamydomonas reinhardtii, Cyanophora paradoxa, Cynara cardunculus var altilis, Emiliana huxleyi, Euglena gracilis, Gonyaulax polyedra, Heterosigma akashiwo, Micromonas pusila, Porphyra sp. Spinacia oleracea, Synechococcus PCC 7942, Thalassiosira pseudonana
Predicted reactivity		Alpha proteobacteria, Algae (brown and red), Beta-proteobacteria, Dicots, Conifers, Cryptomonad, Cyanobacteria, Gamma-proeobacteria, Liverworts, Mosses, Prochlorophytes, Pelwitschia
Not reactive in
Additional information		Quantitative western blot: detailed method description, video tutorial Discussion over some critical aspects of quantitative western blot: Heidebrecht et al. (2009). Improved semiquantitative Western blot technique with increased quantification range. J. Immunological Methods. 35:40-48.
Selected references		Sorrentino et al. (2018). Performance of three cardoon cultivars in an industrial heavy metal-contaminated soil: Effects on morphology, cytology and photosynthesis. J Hazard Mater. 2018 Jun 5;351:131-137. doi: 10.1016/j.jhazmat.2018.02.044. 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.

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 the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample.

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example

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