RbcL antibody and protein standard: Please store at -20°C (6 months) 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.
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 34 assays (standard curve: 0.0625 pmoles, 0.125 pmoles, 0.25 pmoles)2 x 2 ml of 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)2 x 10 µl of AS09 602, Goat anti-rabbit IgG (H&L), HRP conjugated(amount enough for 50-100 Western blots)1 X 10 ml of AS16 ECL-N-10, AgriseraECL Bright 10 ml (5 ml of component A + 5 ml of component B, trial pack)
Quantitative western blot: detailed method description, video tutorialDiscussion over some critical aspects of quantitative western blot: Heidebrecht et al. (2009). Improved semiquantitative Western blot technique with increased quantification range. J. Immunological Methods. 35:40-48.
Defez et al. (2019). Bacterial IAA-Delivery into Medicago Root Nodules Triggers a Balanced Stimulation of C and N Metabolism Leading to a Biomass Increase. Microorganisms. 2019 Sep 29;7(10). pii: E403. doi: 10.3390/microorganisms7100403.Sorrentino et al. (2018). Performance of three cardoon cultivars in an industrial heavy metal-contaminated soil: Effects on morphology, cytology and photosynthesis. J Hazard Mater. 2018 Jun 5;351:131-137. doi: 10.1016/j.jhazmat.2018.02.044.Mota et al. (2015). Effects of heavy metals on Cyanothece sp. CCY 0110 growth, extracellular polymeric substances (EPS) production, ultrastructure and protein profiles. J Proteomics. 2015 Apr 29;120:75-94. doi: 10.1016/j.jprot.2015.03.004.Thamatrakoln et al. (2013). Death-specific protein in a marine diatom regulates photosynthetic responses to iron and light availability. Proc Natl Acad Sci U S A. 2013 Dec 10;110(50):20123-8. doi: 10.1073/pnas.1304727110. Supplemental material describes western blot quantification method.
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.
Host Animal:
Rabbit, Secondary antibody: Goat,
Species Reactivity:
Algae, Cyanobacteria, Higher plants
Immunogen:
KLH-conjugated syntetic peptides for respective antibodies, see product info sheets
50 µl of respective antibody, 100 µl of each protein standard, 2 x 10 µl of secondary antibody, 10 ml of ECL reagent,
Estimation of PSI to PSII ratio can be done using quantitative western blot technique using anti-PsaC (PSI) and PsbA (PSII) antibodies. References: Brown et al. (2007). Resource dynamics during infection of Micromonas pusilla by virus MpV-SP1. Environmental Microbiology 9(11): 2720-2727. Brown et al. (2008). Flux capacities and acclimation costs in Trichodesmium from the Gulf of Mexico. Marine Biology 154 (3): 413-422.
Selected references:
Abramson (2018). CARBON PARTITIONING IN ENGINEERED CYANOBACTERI UM FOR THE STUDY OF FEEDBACK INHIBITION OF PHOTOSYNTHESIS. Michigan State University, ProQuest Dissertations Publishing, 2018. 10826228.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)Bouchard et al. (2006) UVB effects on the photosystem II-D1 protein of phytoplankton and natural phytoplankton communities. Photochem and Photobiol 82: 936-951.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.
Special application note:
Product information - Primary antibodies:Product number:Product name:Reconstitution: Recommended dilution:AS03 037Rabbit Anti-RbcL Global antibodyFor reconstitution see lable on respective tube.1:5000-10 000 with ECLAS10 939Rabbit Anti-PsaC Global antibodyFor reconstitution see lable on respective tube.1:1000 with ECLAS05 084Rabbit Anti-PsbAGlobal antibodyFor reconstitution see lable on respective tube.1:10 000 with ECL* All primary antibodies in this kit are raised in rabbits.Product information - Protein standards:Product number:Product name:Concentration:Size:Western Blot - Positive Control:AS01 016SPsbA *0.25 pmol/ ?l41.5 kDa#To generate a standard curve, 3 loads are suggested (0.5, 2 and 4 ul). For most applications a sample load of 0.2 ug of chlorophyll will give a PsbA signal in this range.A 2 ul load is optimal for most chemiluminescent detection systems to use as a positive control.AS01 017SRbcL *0.15 pmol/ ?l52.7 kDaTo generate a standard curve, 3 loads are suggested (0.5, 2 and 4 ul). For most applications a sample load of 0.2 ug of chlorophyll will give a RbcL signal in this range.A 2 ul load is optimal for most chemiluminescent detection systems to use as a positive control.AS04 042SPsaC *0.15 pmol/?l11.5 kDa#To generate a standard curve, 3 loads are suggested (0.5, 2 and 4 µl). For most applications a sample load of 0.2 µg of chlorophyll will give a PsaC signal in this range.A 2 ul load is optimal for most chemiluminescent detection systems as a positive control.*These proteins are larger than a respective native protein due to the addition of His-tag* For reconstitution of standards see lable on respective tube.Product information - Secondary antibody:AS09 602-trial Goat anti-Rabbit IgG (H&L), HRP conjugated, 20 µl (2x10 µl)Product information - ECLreagent:AS16 ECL-N-10 AgriseraECL Bright (10 ml trial pack)Educational information about Quantitative western blot can be found here: detailed method description, video tutorial
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