Rubisco quantitation kit (Western blot)

AS09 409 | Clonality: Polyclonal | Host: Rabbit

This set enables quantification of Rubisco using Western blot in samples from a range of various species including dicots, monocots, algae, cyanobacteria. The set contains:
Most published anti-RbcL antibody (AS03 037)
Agrisera Protein Extraction Buffer (PEB)
High titer matching secondary antibody (AS09 602)
Chemiluminescent detection reagent of mid picogram range

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.

Product Information

Immunogen KLH-conjugated synthetic peptide was used to elicit anti-RbcL antibody. No baculovirus was used in production of this product.

Host Primary antibody: Rabbit. Secondary antibody: Goat.

Clonality Polyclonal

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)

5 x 10 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 (5 ml of component A + 5 ml of component B, trial pack)

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)

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)

Reactivity

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
Species of your interest not listed? Contact us

Application examples

Application example

example

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.

Agrisera Western Blot protocol and video tutorials

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.

Related products

Related products Rubisco/Carbon metabolism antibody collection

Background

Product citations

Selected references Defez et al. (2019). Bacterial IAA-Delivery into Medicago Root Nodules Triggers a Balanced Stimulation of C and N Metabolism Leading to a Biomass Increase. Microorganisms. 2019 Sep 29;7(10). pii: E403. doi: 10.3390/microorganisms7100403.
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.