RbcL | Rubisco protein standard

220 €

AS01 017S | protein standard for quantitation and positive control

PRODUCT INFORMATION IN PDF

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 a standard: RbcL protein purified from spinach.

immunogen

antibody format

does not apply

quantity

250 µl

lyophilized, for reconstitution add 225 µl of milliQ water per each tube

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

collection of other protein standards

AS01 017 | anti-RbcL Type I hen global antibody

AS03 037 | anti-RbcL Type I and II rabbit global antibody

collection of other global antibodies

collection of antibodies to photosythetic proteins

additional information

The RbcL protein standard can be used in combination with Agrisera global anti-RbcL antibodies to quantitate RbcL from a wide range of species. Global antibodies are raised against highly conserved amino acid sequences in the RbcL protein.

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 0.2μg of chlorophyll will give a RbcL signal in this range. positive control: a 2μl 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 does not require heating before loading on the gel.
expected \| apparent MW		52.7 kDa
confirmed reactivity
predicted reactivity
not reactive in		no confirmed exceptions from predicted reactivity currently known
additional information		Concentration: after adding 225 µl of 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), 50mM DTT. Rubisco protein was purified directly from plant tissue. Please, use the 55 kDa size of RbcL for calculations. The pmoles in the standard refer to pmoles of rbcL monomers.
selected references		Losh et al. (2012). Modest increase in the C:N ratio of N-limited phytoplankton in the California Current in response to high CO2. Marine Ecology 468:31-42.

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 select 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 select 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 applications or usage on species others than stated above Agrisera offers a payment-after-testing option. To learn more about this or for any questions on this product, please use the LiveChat option in the left menue bar or contact us at support@agrisera.com

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