AS05 085 Anti-AtpB | Beta subunit of ATP synthase

Immunogen:	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
Host:	Rabbit
Clonality:	Polyclonal
Purity:	Serum
Format:	Lyophilized
Quantity:	50 µl
Reconstitution:	For reconstitution add 50 µl of sterile water
Storage:	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.
Tested applications:	Blue Native-PAGE (BN-PAGE), Immunofluorescence (IF), Ultrastructure Expansion Microscopy (U-ExM), Western blot (WB)
Recommended dilution:	1 : 100 (IF), 1 : 5000 (BN-PAGE), 1: 2500 (U-ExM), 1 : 2000-1 : 5 000 (WB)
Expected | apparent MW:	53.9 kDa (Arabidopsis thaliana), 51.7 kDa (Synechocystis PCC 6803), 53.7 kDa (Spinacia oleracea)

Confirmed reactivity:	Arabidopsis thaliana, Bacillus cereus, Bryopsis corticulans, Camelina sinensis, Chlamydomonas reinhardtii, Chlorella vulgaris, Chromochloris zofingiensis, Cyanidioschyzon merolae, Dionaea muscipula, Echinochloa crus/galli,  Escherichia coli, Gossypium hirsutum, Helicobacter pylori, Hordeum vulgare, Gladieria sulphuraria, Glycine max, Lycopersicum esulentum, Moniliophthora perniciosa, Nannochloropsis salina, Neochloris oleoabundans (chlorophyta), Nicotiana bentamiana, Nicotiana tabacum, Oryza sp. (roots, leafs, pollen), Ostreococcus tauri, Pheodactylum tricornutum CCAP 1055/1, Pisum sativum, Plasmodium berghei, Populus sp., Robinia pseudoacacia, Scaphoideus titanus, Selaginella martensii, Setaria viridis, Solanum lycopersicum, Spinacia oleracea, Tetrabaena socialis, Toxoplasma gondii, Zea mays   Animal tissues from: cow, chicken, pig, rat, salmon, seal, Locusta migratoria
Predicted reactivity:	Acinetobacter baumannii, Algae, Bacillus subtilis, Brassica napus, Cannabis sativa, Clostridioides difficile, Cyanobacteria, E.coli K-12, Eragrostis tef, Galdieria sulphuraria, Heliomicrobium modesticaldum Ice1, Manihot esculenta, Nicotiana plumbaginifolia, Saccharomyces cerevisiae, Salmonella typhimurium, Trichodesmium erythraeum, Triticum aestivum, Vitis vinifera, Zosteria marina, Yrsinia sp. Species of your interest not listed? Contact us
Not reactive in:	Archeal V-type ATP synthase

2 µg of total protein extracted with PEB (AS08 300) from  leaf tissue of (1) Arabidopsis thaliana, (2) Spinacia oleracea, (3) Lycopersicon esculentum, (4) Glycine max, (5) Populus sp., (6) Zea mays and (7) Hordeum vulgare were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to nitrocellulose. In parallel a dilution row (a-g: 10 - 5 - 2.5 - 1.25 - 0.63 - 0.32 - 0.16 µg protein/lane) from sample 1 (Arabidopsis) was processed. Filters were blocked 1h with 2% low-fat milk powder in TBS-T (0.1% TWEEN 20) and probed with anti-AtpB (AS08 085, 1:5000, 1h) and secondary anti-rabbit (1:10000, 1 h) antibody (HRP conjugated, recommended secondary antibody AS09 602) in TBS-T containing 2% low fat milk powder. Antibody incubations were followed by washings in TBS-T (15, +5, +5, +5 min). All steps were performed at RT with agitation. Signal was detected with chemiluminescent substrate, using a Fuji LAS-3000 CCD (300s, standard sensitivity). 

 

2 µg of total protein from (1) cow muscle, (2) chicken muscle, (3) pig muscle, (4) rat liver, (5) salmon muscle, (6) seal muscle, (8) Arabidopsis thaliana, (9) Zea mays extracted with Protein Extration Buffer, PEB (AS08 300) and separated on  4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% blocking reagent  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: 50 000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (Agrisera anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:50 000 in 2% blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with chemiluminescent detection reagent according to the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad). Exposure time was 30 seconds.

M - molecular weight marker

 

Reactant: Oryza sativa (Asian rice)

Application: Western Blotting

Pudmed ID: 26552588

Journal: Sci Rep

Figure Number: 6A

Published Date: 2015-11-10

First Author: Dixit, G., Singh, A. P., et al.

Impact Factor: 4.13

Open Publication

Western blot analysis of selected candidate proteins (AtpB, 2Fe-2S, GLN2, Psb R, PRK, ALD) with their corresponding molecular weight in rice leaves during As stress under various S regimes (A); RuBisCo large subunit a band in coomassie blue staining (CBB) SDS gel served as a loading control (B), which used for normalization of detected proteins in densitometry studies (C) fold change indicated with respect of control samples.

 

Reactant: Nicotiana tabacum (Common tobacco)

Application: Western Blotting

Pudmed ID: 28180288

Journal: J Exp Bot

Figure Number: 5A

Published Date: 2017-02-01

First Author: Schöttler, M. A., Thiele, W., et al.

Impact Factor: 6.088

Open Publication

Immunoblot analysis of photosynthetic complex accumulation in wild-type tobacco and the two ?psaI lines grown under low, intermediate, and high-light conditions. Because the accumulation of most tested proteins was highest under high-light conditions, lanes one to three contain samples diluted to 25%, 50%, and a 100% sample of wild-type tobacco grown under high-light conditions, to allow for semi-quantitative determination of changes in protein abundance. Lanes four and five contain the two transplastomic lines grown at 1000 µE m?2 s?1. Lanes six to eight contain wild-type tobacco and the mutants grown at intermediate light intensities, and lanes nine to eleven contain samples grown at low light intensities. For PSII, the accumulation of the essential subunits PsbB (CP43) and PsbD (D2) and the LHCB1 antenna protein were determined, while for the cytochrome b6f complex, the accumulation of the essential redox-active subunits PetA (cytochrome f), PetB (cytochrome b6), and PETC (Rieske FeS protein) was tested. AtpB was probed as an essential subunit of the chloroplast ATP. For PSI, in addition to the three essential plastome-encoded subunits PsaA, PsaB, and PsaC, the accumulation of the nuclear-encoded subunits PSAD, PSAH, PSAK, PSAL, and PSAN and of the four LHCI proteins (LHCA1, LHCA2, LHCA3, LHCA4) was determined. Finally, we examined the accumulation of Ycf4, the chloroplast-encoded PSI-biogenesis factor encoded in the same operon as PsaI, and the nuclear-encoded assembly factor Y3IP1.

 

Reactant: Algae

Application: Western Blotting

Pudmed ID: 29460179

Journal: Planta

Figure Number: 7A,B,C

Published Date: 2018-06-01

First Author: Giovagnetti, V., Han, G., et al.

Impact Factor: 3.95

Open Publication

Western blot analysis of PsbS and LHCSR proteins from dark-adapted (D, 30 min), light-treated (L, 2500 µmol photons m?2 s?1, 30 min) and post-dark-recovery chloroplasts (R, 1 h) isolated from B. corticulans. Samples were analysed by immunoblotting with an anti-PsbS (a), anti-LHCSR3 (b) and anti-LHCSR1 antibodies (c). Control samples are dark-adapted S. oleracea (a) and C. reinhardtii chloroplasts (b, c). Note that B. corticulans and S. oleracea samples have been probed against anti-PsbS antibody in the same membrane. The separation between lanes is only due to the presence of different samples between those presented of S. oleracea and B. corticulans (a). The ? subunit of ATP synthase (ATP-B) was used as loading control. 10 µg of Chl was loaded in each lane

Additional information:

The anti-AtpB antibody will detect the mitochondrial form of the F1 ATP  synthase subcomplex, as well as the chloroplastic CF1 Atp Synthase, and most known bacterial F-type Atp Synthases. Peptide used for antibody production is located in a beta sheet, which is partly exposed near the surface of the AtpB protein. Anti-AtpB antibody was used as a loading control in Chlamydomonas reinhardtii and Synechocystis sp. PCC6803. This product can be sold containing proclin if requested

Additional information (application):

Blue Native gel electrophoresis (BN-PAGE) has been performed on samples solubilized with digitonin (4:1) and loaded at 100 µg/well. Gel thickness was 2 mm with 4.5-16 % gradient.   Antibody is recognizing mitochondrial form of AtpB Subota el. al (2011). This antibody can be used as a loading control for bacteria, Bacillus cereus.

ATP synthase is the universal enzyme that synthesizes ATP from ADP and phosphate using the energy stored in a transmembrane ion gradient.

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Agrisera Western Blot protocol and video tutorials

Protocols to work with plant and algal protein extracts

 

Protein Quantitation in plant and algal samples using Agrisera global antibodies 

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.

 

2 µg of total protein from (1-7) Prochlorococcus sp. extracted with Protein Extration Buffer, PEB (AS08 300), (8-11) Rubisco protein standard (AS01 017S) were separated on  4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer for 1h/RT with agitation. Blots were incubated in the primary antibody at a dilution of 1: 20 000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:50 000 for 1h/RT with agitation. The blots were washed as above and developed for 5 min with ECL detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad). There is a low level of a known RbcL cleavage product below the main band in some of the Prochlorococcus samples.

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

Recommended chemiluminescent detection reagent: AgriseraECLBright