AtpB | Beta subunit of ATP synthase (chloroplastic + mitochondrial) (chicken antibodies)
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KLH-conjugated synthetic peptide derived from available plant, algal (chloroplastic and mitochondrial) and bacterial sequences of beta subunits of F-type ATP synthases, including Arabidopsis thaliana chloroplastic ATP synthase subunit beta UniProt: P19366, TAIR: AtCg00480 and Arabidopsis thaliana mitochondrial ATP synthase subunit beta-1, UniProt: P83483, TAIR: At5g08670 as well as Chlamydomonas reinhardtii, UniProt: P06541 and A8IQU3
53.9 kDa (Arabidopsis thaliana), 51.7 kDa (Synechocystis PCC 6803), 53.7 kDa (Spinacia oleracea)
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10 µg of total protein from samples such as beef muscle (1), rat liver (2), Arabidopsis thaliana leaf (3), Hordeum vulgare leaf (4), Synechocystis PCC 6803 total cell (5), Synechococcus PCC 7942 total cell (6) were extracted with 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 70C for 5 min and keep on ice before loading. Protein samples were separated on Bolt 4-12% Tris-Bis gel (Invitrogen) LDS-PAGE and blotted for 1h to 1.5h on PVDF using Bolt Mini Blot Module. Blot was blocked immediately following transfer in 2% blocking reagent (GE RPN 2125; Healthcare) 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. Blot was incubated in the primary antibody at a dilution of 1: 5 000 (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. Blot was incubated in secondary antibody (anti-chicken IgY horse radish peroxidase conjugated) diluted to 1:20 000 in blocking reagent for 1h at room temperature with agitation. The blot was washed as above. Signals in the blot were detected using Lumigen ECL Ultra Reagent (Lumigen TMA-6, Lumigen), and visualized using the Molecular Imager VersaDoc MP 4000 System (Bio-Rad) and Quantity One software (Bio-Rad). Exposure time was 30 seconds.
Reactant: Chlamydomonas reinhardtii (Green Alga)
Application: Western Blotting
Pudmed ID: 25653846
Figure Number: 1A
Published Date: 2015-02-06
First Author: Johnson, E. A. & Lecomte, J. T.
Impact Factor: NoneOpen Publication
Purification of THB1 fromC. reinhardtii cell culture.Protein samples from different stages of purification were analyzed by electrophoresis. (A) Samples of protein extracts before and after lysis ofC. reinhardtii cells with liquid nitrogen. Proteins separated on 16.5% Tris-tricine gel then transferred to nitrocellulose and immunostained with antibodies against proteins localized to different cellular compartments. Lysis by liquid nitrogen enriches the lysate with soluble proteins without enrichment of proteins found in major algal organelles. Histone H3 protein is located in the nucleus, nitrate reductase is a soluble cytosolic protein and the ATP synthase ? subunit is part of the thylakoid membrane of the chloroplast. (B) Samples from different steps in the purification procedure were separated by electrophoresis. Following separation, the proteins within the gel were visualized using silver stain. (C) Proteins prepared identically to those detected in panel B were transferred to nitrocellulose followed by immunostaining with polyclonal antibodies specific for THB1. In addition to the protein samples, a lane was used for molecular weight markers (Spectra LR, ThermoScientific). The numbers indicated between the panels represents location of the markers (kDa molecular mass). WC, whole cell protein extract. Lys, protein extract lysate, and Pel, protein pellet following liquid nitrogen fracturing. QFF, concentrated sample following anion exchange chromatography. S75, concentrated sample following separation on the Superdex 75 column.
Results of immunogold studies using anti-AtpB antibody are published in Andersson et al. (2009).
ATP synthase is the universal enzyme that synthesizes ATP from ADP and phosphate using the energy stored in a transmembrane ion gradient.
Nelson et al. (2019). Protein lysine methylation contributes to modulating the response of sensitive and tolerant Arabidopsis species to cadmium stress. doi: 10.1111/pce.13692.
Gellért et al. (2018). A single point mutation on the cucumber mosaic virus surface induces an unexpected and strong interaction with the F1 complex of the ATP synthase in Nicotiana clevelandii plants. Virus Res. 2018 Jun 2;251:47-55. doi: 10.1016/j.virusres.2018.05.005. (immunogold)
Quesada et al. (2011). Arabidopsis RUGOSA2 encodes an mTERF family member required for mitochondrion, chloroplast and leaf development. Plant J. Nov;68(4):738-53. doi: 10.1111/j.1365-313X.2011.04726.x. Epub 2011 Sep 13.
Andersson et. al (2009). Co-localization of P-glycerate kinase, P-ribulokinase, ADP-glucose pyrophosphorylase and Rubisco activase with CF1 in pea leaf chloroplasts. Plant Science 177:136-14. (immunologold)
Morash et al. (2007). Macromolecular dynamics of the photosynthetic system over a seasonal developmental progression in Spartina alterniflora. Canadian J. of Botany, 2007, 85(5): 476-483, 10.1139/B07-043.
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