PetC | Rieske iron-sulfur protein of Cyt b6/f complex, protein standard

Product no: AS08 330S

AS08 330S  |  Recombinant protein standard for quantitation

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  • Product Info
  • Format: Lyophilized
    Quantity: 250 l
    Reconstitution: For reconstitution add 225 l 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 -20C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
    Tested applications: Western blot (WB)
    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 PsbA signal in this range.

    Positive control:a 2μl load per well is optimal for most chemiluminescent detection systems.

    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: 33 kDa (larger than native protein due to the addition of His-tag), In most gel systems, PetC protein migrates at 23 kDa
  • Additional Information
  • Additional information:

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

    Quantitative western blot:  detailed method description, video tutorial

    Additional information (application):

    Concentration: after adding 225  µl of milliQ water final concentration of the standard is 0.15 pmol/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.

    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.

  • Background
  • Background:

    Rieske Iron-Sulfur Protein (Q9ZR03)is located in chloroplast thylakoid membrane as a component of cytochrome b6-f complex, which mediates electron transfer between photosystem II (PSII) and photosystem I (PSI), cyclic electron flow around PSI, and state transitions. Alternative names: Rieske iron-sulfur protein, RISP, ISP, plastohydroquinone:plastocyanin oxidoreductase iron-sulfur protein, proton gradient regulation protein 1

    This is a recombinant protein standard, source: Synechocystis PCC 6803.

  • Product Citations
  • Selected references: Pipitone et al. (2021). A multifaceted analysis reveals two distinct phases of chloroplast biogenesis during de-etiolation in Arabidopsis. Elife. 2021 Feb 25;10:e62709. doi: 10.7554/eLife.62709. PMID: 33629953; PMCID: PMC7906606.
    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.
    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.
  • Protocols
  • Agrisera Western Blot protocol and video tutorials




    Rubisco quantitation in plant and algal samples using Agrisera anti-petC global antibody and PetC protein standard

    Methodology: Plant samples are generally ground with liquid nitrogen in a mortar and pestle. The resulting powder is transferred to a plastic tube. Algal samples can be either concentrated by centrifugation or, preferably, by filtration onto glass fiber filters. Solubilization is performed in Agrisera protein extraction buffer (PEB, AS08 300) containing 0.1mg/mL PefaBloc SC (AEBSF) protease inhibitor (Roche). Disruption is most optimally obtained through flash freezing of the sample in liquid nitrogen alternated with thawing by sonication with a microtip. This process can be repeated depending on the toughness of the sample. The sample is adjusted to 50 mM dithiothreitol and heated to 70°C for 5 minutes. Samples are cooled and centrifuged briefly prior to electrophoresis.

    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.

    Electrophoresis and Immunoblotting: Once solubilized, the proteins can be separated electrophoretically in a number of systems. We obtain optimal results with the Invitrogen NuPAGE gel system using Bis-Tris 4-12% gradient gels. Proteins are separated in MES SDS running buffer according to the manufacturer’s recommendations at 200 V for 35 minutes. The gels are transferred to PVDF in the same apparatus, the SureLock XCell blot module, for 60 minutes at 30 V for a single gel or 80 minutes for a pair. Following transfer the blots are blocked with non-fat dry milk up to 10 % in TBS-T, for 1 h/RT with gentle agitation. The blot is incubated with primary antibody, usually at 1:25 000 to 1:50 000, for 1 h/RT (if extreme femtogram detection reagents are used) or in lower primary antibody dilution for less sensitivie reagents (mid picogram and lower).

    For quantitation a relatively high primary antibody: target protein ratio gives more reliable results than immunoblots at low ratios of primary antibody:target protein.

    The blot is washed extensively in TBS-T (twice briefly, once for 15 minutes and three times for five minutes). The blot is incubated with secondary antibody, for example goat anti-rabbit IgG horse radish peroxidase conjugated, AS09 602 (Agrisera), for 1h/RT. The blot is washed as above and developed with ECL detection reagents.

    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.


    References:

    MacKenzie et al  (2005). Large reallocations of carbon, nitrogen and photosynthetic reductant among phycobilisomes, photosystems and Rubisco during light acclimation in Synechococcus elongatus are constrained in cells under low environmental inorganic carbon. Arch of Microbiol. 183: 190 - 202.
    Bouchard et al. (2006) UVB effects on the photosystem II-D1 protein of phytoplankton and natural phytoplankton communities. Photochem and Photobiol 82: 936-951. 
    Morash
    et al. (2007) Macromolecular dynamics of the photosynthetic system over a seasonal developmental progression in Spartina alterniflora. Can J. of Bot. 85: 476-483(8)

    Recommended secondary antibodies: goat anti-rabbit HRP conjugated, goat anti-rabbit ALP conjugated


    Recommended chemiluminescent detection reagent: AgriseraECLBright

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Accessories

AS08 330 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, B. rapa subsp. chinensis, C. reinhardtii, E. crus-galli, Euglena sp., H. pluvialis, N. tabacum, P. miliaceum, P. sativum, S. oleracea, Synechococcus PCC 7942, Synechocystis sp. PCC 6803, Thalassiosira guillardii, Z. mays

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AS09 602 |  Clonality: Polyclonal | Host: Goat | Reactivity: Rabbit IgG (H&L)

10 % until end of 2023. Use discount code: Conj10

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AS09 607 |  Clonality: Polyclonal Host: Goat Reactivity: Rabbit IgG (H&L)

10 % until end of 2023. Use discount code: Conj10

206 €
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