PsbA | D1 protein of PSII positive control/quantitation standard

The psbA gene has been cloned from many species of plants, green algae, and cyanobacteria. The psbA gene is located in the chloroplast genome and encodes for the D1 protein, a core component of Photosystem II. PsbA/D1 is rapidly cycled under illumination in all oxygenic photobionts. Tracking PsbA pools using the Global PsbA antibody can show the functional content of Photosystem II in a wide range of samples.This is a recombinant protein standard, source: Synechocystis PCC 6803.

Standard curve: 3 loads are recommended (0.5, 2 and 4 μl) .For most applications a sample load of 0.2 μg of chlorophyll will give a PsbA signal in this range.Positive control: a 2 μl load per well is optimal for most chemiluminescent detection systems.Non-disulphie dependent dimers and complexes can be also detected using standard western blot methods with more sensitive detection reagents as ECL Advance or West Pico when loading per well more standard than recommended. They have not been included in the standard calibration.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.

For reconstitution, check the label on the tube. Please notice that this product contains 10% glycerol and might appear as liquid but is provided lyophilized

The standard has an actual MW of 41.5 kDa, The presence of a His6 tag causes it to run ~1.7 kDa higher on the gel than the native protein, Note that in most systems, PsbA migrates with an apparent MW of between 30 and 35 kDa.

Concentration:after adding 95 µl of sterile milliQ water final concentration of the standard is 0.25 pmoles/µLProtein 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 standardis stabilized and readyand 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.

The PsbA protein standard can be used in combination with global anti-PsbA antibodies to quantitate PsbA from a wide range of species. Global antibodies are raised against highly conserved amino acid sequences in the PsbA protein.Quantitative western blot: detailed method description, video tutorialThe goals when doing quantitative work:The sample PsbA must fall somewhere between the upper and lower standard loads. There should be at least 3 points on the standard curve.if possible, try to make the entire range of the curve around one order of magnitude or less (as in the application example) .if possible, load <5µg total sample protein.1pmol of PsbA standard is a strong load for chemiluminescence, but may be appropriate for the less sensitive reagents, for example alkaline phosphatase.

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.Fernández-González et al. (2020) . Effects of Temperature and Nutrient Supply on Resource Allocation, Photosynthetic Strategy, and Metabolic Rates of Synechococcus Sp . J Phycol . 2020 Mar 4. doi: 10.1111/jpy.12983. Levitan et al. (2019) . Structural and functional analyses of photosystem II in the marine diatom Phaeodactylum tricornutum. Proc Natl Acad Sci U S A. 2019 Aug 27;116 (35) :17316-17322. doi: 10.1073/pnas.1906726116.Ryan-Keogh et al. (2018) . Seasonal regulation of the coupling between photosynthetic electron transport and carbon fixation in the Southern Ocean. Limnology and Oceanography.Yuan et al. (2018) . Combined effects of ocean acidification and warming on physiological response of the diatom Thalassiosira pseudonana to light challenges. Mar Environ Res. 2018 Apr;135:63-69. doi: 10.1016/j.marenvres.2018.01.016.

Store lyophilized/reconstituted at -20°C; 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.