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RbcL | Rubisco positive control/quantitation standard

169 €
Buy 2 items of this product for 140 €/each
Buy 3 items of this product for 120 €/each

AS01 017S  |  protein standard for quantitation and positive control

When you buy this product you get a 50% discount on other protein standards.
Follow this link to a list of discounted standards.
To get this discount please send your order to orders@agrisera.com

PRODUCT INFORMATION IN PDF

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AS01 017S

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product information
Background

Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. It is one of the most abundant proteins on Earth and its homology has been demonstrated from purple bacteria to flowering plants.

Source of Rubisco standard: Rubisco protein was purified directly from a plant tissue - spinach. 

Host
Clonality
Clone
Purity
Format
Quantity 100 ĩl
Reconstitution For reconstitution add 90 ĩl of milliQ water.
Please notice that this product contains 10% glycerol and might appear as liquid but is provided lyophilized.
Storage

store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.

Tested applications western blot (WB)
Related products

Primary antibodies matching Rubisco standard are:

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)

AS01 017  | anti-RbcL | Rubisco large subunit, form I, chicken antibody

AS15 2994 | Rubisco ELISA quantitation kit 

matching secondary antibody

collection of other protein standards

collection of other global antibodies

Plant and algal protein extraction buffer

Additional information

The RbcL protein standard can be used in a combination with Agrisera global antibiodies (AS01 017 from chicken or AS03 037 from a rabbit) to quantitate RbcL from a wide range of species. Global antibodies are raised against highly conserved amino acid sequence. This standard is also included in following kits: Educational antibody kit - photosynthesisPhotosynthesis Tool Kit - quantitation, Rubico quantitation kit,

Quantitative western blot:  detailed method description, video tutorial

application information
Recommended dilution

Standard curve: 3 loads are recommended (0.5, 2 and 4μl).
For most applications a sample load of 0.2 μg of chlorophyll/well will give a RbcL signal in this range.

Positive control: a 2 μl load per well is optimal for most chemiluminescent detection systems. Higher standard concentration needs to be used to allow detection by Coomasie stains. Such gels with higher standard concentration can not be used for quantitation using chemiluminescence.

This standard is stabilized and ready and does not require heating before loading on the gel.

Please note that this product contains 10% glycerol and might appear as liquid but is provided lyophilized. Allow the product several minutes to solubilize after adding water. Mix thoroughly but gently Take extra care to mix thoroughly before each use, as the proteins tend to settle with the more dense layer after freezing.

Expected | apparent MW

52.7 kDa

Confirmed reactivity
Predicted reactivity
Not reactive in
Additional information

Concentration: after re-constitution with sterile milliQ water final concentration of the standard is 0.15 pmoles/µl

Protein standard buffer composition: Glycerol 10%, Tris Base 141 mM, Tris HCl 106 mM, LDS 2%, EDTA 0.51 mM, SERVA® Blue G250 0.22 mM, Phenol Red 0.175 mM, pH 8.5, 0.1mg/ml PefaBloc protease inhibitor (Roche), 50 mM DTT.

This standard is ready-to-load and does not require any additions or heating. It needs to be fully thawed and thoroughly mixed prior to using. Avoid vigorous vortexing, as buffers contain detergent. Following mixing, briefly pulse in a microcentrifuge to collect material from cap.
This standard is stabilized and ready and does not require heating before loading on the gel.
Please note that this product contains 10% glycerol and might appear as liquid but is provided lyophilized. Allow the product several minutes to solubilize after adding water. Mix thoroughly but gently Take extra care to mix thoroughly before each use, as the proteins tend to settle with the more dense layer after freezing.

Please, use the 55 kDa size of RbcL for calculations. The pmoles in the standard refer to pmoles of rbcL monomers.

Selected references
Li et al. (2016). Interactive effects of nitrogen and light on growth rates and RUBISCO content of small and large centric diatoms. Photosynth Res. 2016 Aug 26. [Epub ahead of print]
Young et al. (2015). Antarctic phytoplankton down-regulate their carbon-concentrating mechanisms under high CO2 with no change in growth rates. Marine Ecology Progress Series 532:13-28.
Vandenhecke et al. (2015). Changes in the Rubisco to photosystem ratio dominates photoacclimation across phytoplankton taxa. Photosynth Res. 2015 Apr 11.
Wu et al. (2014). Large centric diatoms allocate more cellular nitrogen to photosynthesis to counter slower RUBISCO turnover rates. Front. Mar. Sci., 09 December 2014 | doi: 10.3389/fmars.2014.00068.
Li
 et al. (2014). The nitrogen costs of photosynthesis in a diatom under current and future pCO2. New Phytol. 2014 Sep 25. doi: 10.1111/nph.13037.

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.


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.


 example of Rubisco quantitation

Quantitation: When quantitated standards are included on the blot, the samples can be quantitated using the available software. Excellent quantitation can be obtained with images captured on the Bio-Rad Fluor-S-Max or equivalent instrument using Bio-Rad QuantityOne software. The contour tool 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. Using above protocol linear standard curves are generated over 1-1.5 orders of magnitude range in target load. It is important to note that immunodetections usually show a strongly sigmoidal signal to load response curve, with a region of trace detection of low loads, a pseudolinear range and a region of saturated response with high loads. For immunoquantitation it is critical that the target proteins in the samples and the standard curve fall within the pseudolinear range. Our total detection range using this protocol spans over 2 orders of magnitude, but the quantifiable range is narrower.
Quantitative western blot:  detailed method description.


||| For other applications or usage on species other than stated above Agrisera offers a payment-after-testing option. To learn more about this or for any questions please use the LiveChat option or contact us at support@agrisera.com