- Model 2 - Quantitation Kit



The Rubisco quantitation kit is suitable for quantitation of Rubisco in plant and algal samplesusing quantitative immunoblotting. It contains anti-RbcL antibody, Rubisco protein standard and theProtein 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.

AS09 409 - clonality:polyclonal - host: rabbit - r eactivity: dicots, monocots, algae, cyanobacteria

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

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 85assays (standard curve: 0.0625 pmoles, 0.125 pmoles, 0.25 pmoles)2 x 2 ml 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)

RbcL antibody and protein standard:
Please stoore 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)

Detecting and quantitating important proteins in plants and other photosynthetic organisms has become more practical and powerful with the application of quantitative immunoblotting using global antibodies which recognize proteins evenly across a wide range of taxa. While many tools are commercially available for the examination of medically relevant proteins in mammals, such tools have been lacking for the study of pressing questions in other organisms. Here we present the advantages of quantitative immunoblotting and the methodology necessary to apply these tools.

Introduction | The exquisite specificity of the antibody antigen interaction has long been exploited by protein biochemists. Antibodies bind to small regions on proteins, termed epitopes, which often consist of only a few amino acids. This binding is highly specific
and of high affinity. As each antibody recognizes only a small part of the whole protein, an immunogenic peptide can be selected using sequence alignments. This short peptide is highly conserved and exclusive to the protein under study. The resulting antibodies are then immunoreactive with the conserved peptide region within intact proteins derived from a number of species. We refer to these as Global Antibodies, and they provide both scientific and practical advantages. In non-medical areas such as plant sciences, comparative analyses across species, is often productive; Global Antibodies permit such cross-taxon studies with level detection efficiencies across wide taxonomic ranges. From a practical standpoint, a Global Antibody functioning across a wide taxonomic range reduces the cost of antibody production and stocking by creating a detection tool useful in a wide range of studies.

The ELISA, or Enzyme Linked ImmunoSorbent Assay, employs specific antibodies in a high through put application for the measurement of specific substances in samples of diverse origins. While some sample and antibody combinations can be optimized for detections using ELISA, others are less successful. This can be the result of antibodies with significant cross reactivities, samples that do not adhere optimally to the plate, a lack of quantitated standards or samples of variable quality that display differential reactivity in ELISA. A similar approach is the quantitative immunoblot. As such, it uses the same principles as the ELISA, a reliance on the highly specific antibody antigen interaction to measure the amount of a given protein in a complex mixture. The major distinguishing feature of this method is the inclusion of a step to electrophoretically separate the proteins in the sample according to their molecular weights. Thus, all samples can be solubilized using a standardized procedure and analyzed concurrently for a number of interesting proteins. The separation step allows the researcher to be more confident that the signal generated actually results from the target protein in question because it is spatially resolved from cross-reactions which would confound an ELISA system.

Limitations of Traditional Techniques | A number of traditional techniques are available for the measurement of different functions or complexes of photosynthetic organisms. These include an array of enzyme and functional assays. A disadvantage of such approaches is that they require individually specialized sample harvesting and preparation techniques, and often specialized equipment or reagents for each assay. Therefore a single sample preparation cannot be used to quantify different protein complexes. Furthermore, many samples are not available in the quantities or preservation states necessary for multiple determinations of different protein complexes.

Advantages of Immunodetections with Global Antibodies | When Global Antibodies are used for quantitative immunodetections even small, mixed samples from various harvesting procedures can be assessed for multiple complexes simultaneously. Since all detections employ a common solubilization protocol as a starting point, comparisons between complexes can be made with greater confidence. Since Global Antibodies recognize short highly conserved regions of the protein targets they can be applied to a very broad range of target organisms. They can also be applied with considerable confidence to uncharacterized or mixed samples. 

Quantitated recombinant protein standards have been generated to be used as a calibration with Global Antibodies. The target peptide antigen is perfectly conserved in these standard proteins, thus they can be applied for comparative quantitation of the target protein from all species.

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