SBPase | Sedoheptulose-1,7-bis phosphatase
AS15 2873 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, Chlamydomonas sp. (strain W80), C. sativus, H. vulgare, O. sativa, Z. mays
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10 µg of total protein from Arabidopsis thaliana leaf (1) , SBPase protein standard AS15 2873S (2) were extracted with Agrisera Protein Extraction Buffer PEB (AS08 300). Samples were diluted with 1X sample buffer (NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM DTT and heat at 70°C for 5 min and keept on ice before loading. Protein samples were separated on 4-12% Bolt Plus gels, LDS-PAGE and blotted for 70 minutes to PVDF using tank transfer. Blots were blocked immediately following transfer in 2% blocking reagent or 5% non-fat milk dissolved in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 10 000 (in blocking reagent) for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed 1x15 min and 3x5 min with TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS09 602, Agrisera) diluted to 1:25 000 in blocking reagent for 1h at room temperature with agitation. The blots were washed as above. The blot was developed for 5 min with chemiluminescent detection reagent of extreme femtogram sensitivity, according the manufacturers instructions. Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 30 seconds.
5 or 10 µg of total protein from Chlamydomonas reinhardtii extracted with 10 mM Tris/HCl pH7.5, 80 mM NaCl, 1 mM EDTA, 1 % (w/v) Glycerol, 1 mM DTT, 1x protease inhibitor cocktail (Roche) and denatured in SDS sample buffer for 5 min. at 95°C were separated on 12 % SDS-PAGE and blotted over night to nitrocellulose using tank transfer. Blots were blocked with 3 % milk; for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 500 in blocking buffer for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 5 min. 5x in TBS-T (0.05 %) at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated from Agrisera AS09 602) diluted to 1:25 000; for 1h at RT with agitation. The blot was washed as above and developed using Luminol-H2O2 and p-Coumaric acid. Exposure time of X-ray film was 1 hour.
Courtesy of Dr. Sebastian Mahlow, Prof. Maria Mittag, Institute of General Botany and Plant Physiology, University of Jena, Germany
Reactant: Chlamydomonas reinhardtii (Green Alga)
Application: Western Blotting
Pudmed ID: 32655601
Journal: Front Plant Sci
Figure Number: 1B
Published Date: 2020-07-14
First Author: Hammel, A., Sommer, F., et al.
Impact Factor: 5.435Open Publication
Generation of Chlamydomonas lines overexpressing SBP1. (A) SBP1 construct used for transformation. The 2172 bp SBP1 ORF (exons shown as black boxes), interrupted by all native SBP1 introns (thin lines), was domesticated to generate a level 0 module for the MoClo strategy. Using MoClo, the SBP1 ORF was equipped with the HSP70A-RBCS2 promotor and the RPL23 terminator (pAR and tRPL23, respectively, white boxes) and with a 3xHA-tag or without a tag (mStop) (gray box), giving rise to two level 1 constructs. These were combined with another level 1 construct containing the aadA gene conferring resistance to spectinomycin (light gray box) to yield the final level 2 constructs for transformation. (B) Screening of transformants overexpressing SBP1. The UVM4 strain was transformed with the level 2 constructs shown in (A). Total cell proteins from 12 spectinomycin resistant transformants recovered with each construct were extracted and proteins corresponding to 1.5 ?g chlorophyll were analyzed by immunoblotting using anti-HA or anti-SBPase antibodies. The transformant number is given on top of the lanes. Transformants exhibiting highest expression levels for SBP1-3HA or SBP1-mStop (red) were used for further analysis.
Hammel et al. (2020) Photosynthesis in Chlamydomonas reinhardtii and Has No Effect on the Abundance of Other Calvin-Benson Cycle Enzymes. Front Plant Sci. 2020 Jun 23;11:868. doi: 10.3389/fpls.2020.00868. PMID: 32655601; PMCID: PMC7324757.
Fukayama et al. (2018). Expression level of Rubisco activase negatively correlates with Rubisco content in transgenic rice. Photosynth Res. 2018 May 30. doi: 10.1007/s11120-018-0525-9.
Li et al. (2018). Comparative proteomic analysis of key proteins during abscisic acid-hydrogen peroxide-induced adventitious rooting in cucumber (Cucumis sativus L.) under drought stress. Journal of Plant Physiology Volume 229, October 2018, Pages 185-194.
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