PEPC | Phosphoenolpyruvate carboxylase

AS09 458 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. comosus, A. thaliana, C. ciliaris, C. gayana, C. velia, H. vulgare, J. curcas, K. prostrata, L. fusca, Lupinus sp. , M. maximus, M. crystallinum, N. tabacum, O. sativa, P. antidotale, P. coloratum, P. strobus, Saccharum spp. hybrid clone C91-301, S. lanata, S. laricifolia, S. bicolor, Synechocystis PCC 6803, Phaeodactylum tricornutum (strain CCAP 1055/1), T. weissfloggi, Z. mays, Z. muelleri

Benefits of using this antibody

Product Information

Immunogen

KLH-conjugated synthetic peptide well conserved PEPC1 and sequences from different plant species including Arabidopsis thaliana Q9MAH0, At1g53310 (PEPC 1), Q84VW9, At3g14940 (PEPC 3). The peptide chosen to elicit this antibody is also perfectly conserved in bacterial type of this enzyme NP_177043.2 (PEPC 4).

For Zea mays, the peptide is converved in PEP1 and PEP4 isoforms.

Host Rabbit

Clonality Polyclonal

Purity Immunogen affinity purified serum in PBS pH 7.4.

Format Lyophilized

Quantity 50 µg

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. Please do not re-use this primary antibody solution. In case of cyanobacterial samples there will be no signal in your second incubation.

Tested applications Immunolocalization (IL), Western blot (WB)

Recommended dilution 1 : 500 (IL), 1: 1000 - : 10 000 (WB)

Expected | apparent MW

110 | 105 kDa

Reactivity

Confirmed reactivity Ananas comosus, Arabidopsis thaliana, Cenchrus ciliaris, Chloris gayana, Chromera velia, Cyanthobasis fruticulosa, Hordeum vulgare, Jatropha curcas, Kochia prostrata, Leptochloa fusca, Lupinus sp. , Megathyrsus maximus, Mesembryanthemum crystallinum, Nicotiana tabacum, Oryza sativa, Panicum antidotale, Panicum coloratum, Petrosimonia nigdeensis, Pinus strobus, Saccharum spp. hybrid clone C91-301, Salsola lanata, Salsola laricifolia,Salsola grandis, Salsola tragus Sorghum bicolor, Synechocystis PCC 6803, Phaeodactylum tricornutum (strain CCAP 1055/1), Pinus strobus, Thalassiosira weissfloggi, Zea mays, Zostera muelleri

Predicted reactivity

Cucumis sativus (PEPC1, PEPC2, PEPC3), Flaveria bidentis, Flaveria trinervia, Glycine max, Lupinus albus, Mammillaria thornberi, Manihot esculenta, Manihot obovata, Medicago sativa, Morinda citrifolia, Nannochloropsis gaditana CCMP526, Nopalea gaumeri, Opuntia macbridei, Pachycereus pringlei, Saccharum spp, Solanum tuberosum, Spinacia oleracea, Streptanthus tortuosus, Pachycereus hollianus, Pisum sativa, Phaseolus vulgaris, Populus sp.,Triticum aestivum, algae, diatoms: Thalassiosira pseudonana, other species: Salmonella sp., Schiedea hookeri, Shigella sp. Schiedea sarmentosa, Streptanthus farnsworthianus, Tacinga saxatilis,Yersinia sp. Vibrio sp., Quercus sp.

Species of your interest not listed? Contact us

Not reactive in Methanothermobacter thermautotrophicus

Application examples

Application example

5 µg of total protein from (1) Arabidopsis thaliana leaf extracted with Protein Extration Buffer, PEB (AS08 300), (2) Spinacia oleracea total cell, extracted with PEB, (3) Hordeum vulgare total cell extracted with PEB, (4) Zea mays total cell extracted with PEB, were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% blocking reagent (GE Healthcare) 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 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 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 the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).

Western blot using anti-PEPC antibodies on A.thaliana and Pinus strobus

10 µg of total protein extracted freshly from Arabidopsis thaliana wt leaf tissue (At_nnon-senescent leaves), Arabidopsis thaliana wt leaf tissue (At_s senescent leaves), Pinus strobus needle tissue (PS_0,PS₃₆) with 1 M Tris-HCl, pH 6.8, 10 % SDS, 15 % sucrose, 0.5 DTT and denatured at 75°C for 5 min. were separated on 10 % Bis-Tris Nupage Novex gel (120 V/45 min. using MES buffer system) and blotted 30 min. to PVDF. Blot was blocked with 5 % non-fat milk 45 min./RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 for 1h/RT with agitation in TBS with 2 % non-fat milk or ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice for 10 min. in TBS at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:75 000 in for 1h/RT with agitation. The blot was washed as above and developed using chemiluminescent detection. Exposure time was 40 seconds.

Courtesy of Dr. Christine Yao-Yun Chang and the Ensminger lab, University of Toronto, Canada

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 24723409

Journal: J Exp Bot

Figure Number: 4A

Published Date: 2014-07-01

First Author: Pinto, H., Sharwood, R. E., et al.

Impact Factor: 6.088

Open Publication

Immunoblot analyses of photosynthetic enzymes. Examples of immunoblot analysis for the photosynthetic proteins Rubisco (A), PEPC (B), NADP-ME (C), and PEP-CK (D) extracted from leaves of selected grass species grown at glacial (180 ?l l–1, G) or ambient (400 ?l l–1, A) [CO2].

Reactant: Zea mays (Maize/Corn)

Application: Western Blotting

Pudmed ID: 26208645

Journal: J Exp Bot

Figure Number: 10C

Published Date: 2015-11-01

First Author: Chen, J., Wu, F. H., et al.

Impact Factor: 6.088

Open Publication

Western blot analysis of RuBISCO LSU (A) and PEPC (C) of maize plants. Maize seedlings were pre-treated with 100 µM NaHS for 8 d and then grown in a nutrient solution containing 1 µM Fe(III)-EDTA or 50 µM Fe(III)-EDTA for 12 d. Relative expression level is shown as the ratio of RuBISCO LSU:?-actin (B) and PEPC:?-actin (D) using Quantity One software. Data are presented as means ± SE. Columns labelled with different letters indicate significant differences at P<0.05. –Fe, 1 µM Fe; –Fe+NaHS, seedlings were pre-treated with 100 µM NaHS and then treated with 1 µM Fe; +Fe, 50 µM Fe; +Fe+NaHS, seedlings were pre-treated with 100 µM NaHS and then treated with 50 µM Fe.

Reactant: Echinochloa (Barnyard grass)

Application: Western Blotting

Pudmed ID: 29659931

Journal: J Exp Bot

Figure Number: 5A

Published Date: 2018-05-25

First Author: Sonawane, B. V., Sharwood, R. E., et al.

Impact Factor: 6.088

Open Publication

Immunoblot analysis of photosynthetic enzymes. Immunoblot analysis for the photosynthetic proteins Rubisco, PEPC, PEP-CK, and NADP-ME extracted from leaves of eight C4 grasses belonging to three biochemical subtypes in control (C) or shade (S) environments. Loaded volumes varied between 4 ?l and 15 ?l in order to normalize the protein content to a common leaf area. Because of the small gel size, a limited number of samples (8–9) was loaded on an individual gel. Finally, all immunoblots of the studied protein and species were arranged in a composite figure. For uniform visualization, gamma settings of individual images were adjusted. A protein ladder was used for individual immunoblots; for simplicity, band size is referred to numerically.

Reactant: Oryza sativa (Asian rice)

Application: Western Blotting

Pudmed ID: 30499169

Journal: Mol Plant Pathol

Figure Number: 3A

Published Date: 2019-04-01

First Author: Hui, S., Shi, Y., et al.

Impact Factor: 5.418

Open Publication

Expression patterns of OsImp?1a and OsImp?1b. (A) OsImp?1a and OsImp?1b are nucleocytoplasmic?localized proteins. Total protein, the nucleus?depleted fraction and nucleus?enriched fraction were loaded onto a sodium dodecylsulfate?polyacrylamide gel electrophoresis (SDS?PAGE) gel and subjected to immunoblot analysis. Histone H3 and phosphoenolpyruvate carboxylase (PEPC) were used as nuclear and cytosolic markers, respectively. T, total protein extracts; N, nucleus?enriched fraction; C, nucleus?depleted fraction. (B) Expression of OsImp?1a and OsImp?1b in different tissues. Tissues were collected at the booting stage from IR24. (C) Expression of OsImp?1a and OsImp?1b after infection with Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99.

Additional information

Antibody can be also used following 2D gel electrophoresis

Background

PEPC (phosphoenolpyruvate carboxylase), EC=4.1.1.31, belongs to an enzyme family of carboxy-lyases that is catalyzing adding fo carbon dioxide to phosphoenolpyruvate (PEP) to form oxaloacetate. Alternative names: PEPCase 1, PEPCase 3, PEPC 1, PEPC 3

Product citations

Selected references Durall et al. (2021). Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt. Microb Cell Fact. 2021 Feb 8;20(1):39. doi: 10.1186/s12934-021-01529-y. PMID: 33557832; PMCID: PMC7871529.
Wang et al. (2021). Brassinosteroids inhibit miRNA-mediated translational repression by decreasing AGO1 on the endoplasmic reticulum. J Integr Plant Biol. 2021 May 21. doi: 10.1111/jipb.13139. Epub ahead of print. PMID: 34020507.
Rakhmankulova et al. (2021) Possible Activation of ?3 Photosynthesis in ?4 Halophyte Kochia prostrata Exposed to an Elevated Concentration of ??2. Russ J Plant Physiol 68, 1107–1114 (2021). https://doi.org/10.1134/S1021443721060169
Durall et al. (2020). Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803. Biotechnol Biofuels. 2020 Jan 28;13:16. doi: 10.1186/s13068-020-1653-y.
Kramer et al. (2020). N6?methyladenosine and RNA secondary structure affect transcript stability and protein abundance during systemic salt stress in Arabidopsis. Plant Direct . 2020 Jul 24;4(7):e00239.doi: 10.1002/pld3.239.
Wang et al. (2019). PUB25 and PUB26 Promote Plant Freezing Tolerance by Degrading the Cold Signaling Negative Regulator MYB15.
Hui et al. (2018). TALE-carrying bacterial pathogens trap host nuclear import receptors for facilitation of infection of rice. Mol Plant Pathol. 2018 Nov 30. doi: 10.1111/mpp.12772.
Salesse-Smith et al. (2018). Overexpression of Rubisco subunits with RAF1 increases Rubisco content in maize. Nat Plants. 2018 Oct;4(10):802-810. doi: 10.1038/s41477-018-0252-4.
Bassi et al. (2018). Nitrogen supply influences photosynthesis establishment along the sugarcane leaf. Sci Rep. 2018 Feb 2;8(1):2327. doi: 10.1038/s41598-018-20653-1.
Sonawane et al. (2018). Shade compromises the photosynthetic efficiency of NADP-ME less than PEP-CK and NAD-ME C 4 grasses. J Exp. Botany, doi.org/10.1093/jxb/ery129.
Wen et al. (2017). Possible involvement of phosphoenolpyruvate carboxylase and NAD-malic enzyme in response to drought stress. A case study: A succulent nature of the C4-NAD-ME type desert plant, Salsola lanata (Chenopodiaceae). Functional Plant Biology 44(12), DOI10.1071/FP16430
Jiang et al. (2017). Development of an Efficient Protein Extraction Method Compatible with LC-MS/MS for Proteome Mapping in Two Australian Seagrasses Zostera muelleri and Posidonia australis. Frontiers in Plant Science, doi: 10.3389/fpls.2017.01416.
Liu et al. (2017). Plasma Membrane CRPK1-Mediated Phosphorylation of 14-3-3 Proteins Induces Their Nuclear Import to Fine-Tune CBF Signaling during Cold Response. Mol Cell. 2017 Apr 6;66(1):117-128.e5. doi: 10.1016/j.molcel.2017.02.016.
Ribeiro et al. (2017). Increased sink strength offsets the inhibitory effect of sucrose on sugarcane photosynthesis. J Plant Physiol. 2017 Jan;208:61-69. doi: 10.1016/j.jplph.2016.11.005.
Shen et al. (2016). The existence of C4-bundle-sheath-like photosynthesis in the mid-vein of C3 rice. Rice (N Y). 2016 Dec;9(1):20. doi: 10.1186/s12284-016-0094-5. Epub 2016 May 10.
Ishikawa et al. (2016). NDH-Mediated Cyclic Electron Flow Around Photosystem I is Crucial for C4 Photosynthesis. Plant Cell Physiol. 2016 Aug 6. pii: pcw127. [Epub ahead of print]
Shen et al. (2015). Overexpression of maize phosphoenolpyruvate carboxylase improves drought tolerance in rice by stabilization the function and structure of thylakoid membrane. Photosynthetica, September 2015, Volume 53, Issue 3, pp 436-446.
Foley et. al (2015). Analysis of conglutin seed storage proteins across lupin species using transcriptomic, protein and comparative genomic approaches. BMC Plant Biology 2015, 15:106 doi:10.1186/s12870-015-0485-6.

calculated \| apparent molecular mass [kDa]:	110 \| 105 kDa
Clonality:	Polyclonal
Format:	Lyophilized
Host:	Rabbit
immunogen:	KLH-conjugated synthetic peptide well conserved PEPC1 and sequences from different plant species including Arabidopsis thaliana Q9MAH0, At1g53310 (PEPC 1), Q84VW9, At3g14940 (PEPC 3). The peptide chosen to elicit this antibody is also perfectly conserved in bacterial type of this enzyme NP_177043.2 (PEPC 4). For Zea mays, the peptide is converved in PEP1 and PEP4 isoforms.
Purity:	Immunogen affinity purified serum in PBS pH 7.4.
Quantity:	50 µg
recommended dilution:	1 : 500 (IL), 1: 1000 - : 10 000 (WB)
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. Please do not re-use this primary antibody solution. In case of cyanobacterial samples there will be no signal in your second incubation.
tested applications:	Immunolocalization (IL), Western blot (WB)
All references:	Durall et al. (2021). Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt. Microb Cell Fact. 2021 Feb 8;20(1):39. doi: 10.1186/s12934-021-01529-y. PMID: 33557832; PMCID: PMC7871529. Wang et al. (2021). Brassinosteroids inhibit miRNA-mediated translational repression by decreasing AGO1 on the endoplasmic reticulum. J Integr Plant Biol. 2021 May 21. doi: 10.1111/jipb.13139. Epub ahead of print. PMID: 34020507. Rakhmankulova et al. (2021) Possible Activation of ?3 Photosynthesis in ?4 Halophyte Kochia prostrata Exposed to an Elevated Concentration of ??2. Russ J Plant Physiol 68, 1107–1114 (2021). https://doi.org/10.1134/S1021443721060169 Durall et al. (2020). Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803. Biotechnol Biofuels. 2020 Jan 28;13:16. doi: 10.1186/s13068-020-1653-y. Kramer et al. (2020). N6?methyladenosine and RNA secondary structure affect transcript stability and protein abundance during systemic salt stress in Arabidopsis. Plant Direct . 2020 Jul 24;4(7):e00239.doi: 10.1002/pld3.239. Wang et al. (2019). PUB25 and PUB26 Promote Plant Freezing Tolerance by Degrading the Cold Signaling Negative Regulator MYB15. Hui et al. (2018). TALE-carrying bacterial pathogens trap host nuclear import receptors for facilitation of infection of rice. Mol Plant Pathol. 2018 Nov 30. doi: 10.1111/mpp.12772. Salesse-Smith et al. (2018). Overexpression of Rubisco subunits with RAF1 increases Rubisco content in maize. Nat Plants. 2018 Oct;4(10):802-810. doi: 10.1038/s41477-018-0252-4. Bassi et al. (2018). Nitrogen supply influences photosynthesis establishment along the sugarcane leaf. Sci Rep. 2018 Feb 2;8(1):2327. doi: 10.1038/s41598-018-20653-1. Sonawane et al. (2018). Shade compromises the photosynthetic efficiency of NADP-ME less than PEP-CK and NAD-ME C 4 grasses. J Exp. Botany, doi.org/10.1093/jxb/ery129. Wen et al. (2017). Possible involvement of phosphoenolpyruvate carboxylase and NAD-malic enzyme in response to drought stress. A case study: A succulent nature of the C4-NAD-ME type desert plant, Salsola lanata (Chenopodiaceae). Functional Plant Biology 44(12), DOI10.1071/FP16430 Jiang et al. (2017). Development of an Efficient Protein Extraction Method Compatible with LC-MS/MS for Proteome Mapping in Two Australian Seagrasses Zostera muelleri and Posidonia australis. Frontiers in Plant Science, doi: 10.3389/fpls.2017.01416. Liu et al. (2017). Plasma Membrane CRPK1-Mediated Phosphorylation of 14-3-3 Proteins Induces Their Nuclear Import to Fine-Tune CBF Signaling during Cold Response. Mol Cell. 2017 Apr 6;66(1):117-128.e5. doi: 10.1016/j.molcel.2017.02.016. Ribeiro et al. (2017). Increased sink strength offsets the inhibitory effect of sucrose on sugarcane photosynthesis. J Plant Physiol. 2017 Jan;208:61-69. doi: 10.1016/j.jplph.2016.11.005. Shen et al. (2016). The existence of C4-bundle-sheath-like photosynthesis in the mid-vein of C3 rice. Rice (N Y). 2016 Dec;9(1):20. doi: 10.1186/s12284-016-0094-5. Epub 2016 May 10. Ishikawa et al. (2016). NDH-Mediated Cyclic Electron Flow Around Photosystem I is Crucial for C4 Photosynthesis. Plant Cell Physiol. 2016 Aug 6. pii: pcw127. [Epub ahead of print] Shen et al. (2015). Overexpression of maize phosphoenolpyruvate carboxylase improves drought tolerance in rice by stabilization the function and structure of thylakoid membrane. Photosynthetica, September 2015, Volume 53, Issue 3, pp 436-446. Foley et. al (2015). Analysis of conglutin seed storage proteins across lupin species using transcriptomic, protein and comparative genomic approaches. BMC Plant Biology 2015, 15:106 doi:10.1186/s12870-015-0485-6.
background:	PEPC (phosphoenolpyruvate carboxylase), EC=4.1.1.31, belongs to an enzyme family of carboxy-lyases that is catalyzing adding fo carbon dioxide to phosphoenolpyruvate (PEP) to form oxaloacetate. Alternative names: PEPCase 1, PEPCase 3, PEPC 1, PEPC 3
additional information (application):	Antibody can be also used following 2D gel electrophoresis
Confirmed reactivity:	Ananas comosus, Arabidopsis thaliana, Cenchrus ciliaris, Chloris gayana, Chromera velia, Cyanthobasis fruticulosa, Hordeum vulgare, Jatropha curcas, Kochia prostrata, Leptochloa fusca, Lupinus sp. , Megathyrsus maximus, Mesembryanthemum crystallinum, Nicotiana tabacum, Oryza sativa, Panicum antidotale, Panicum coloratum, Petrosimonia nigdeensis, Pinus strobus, Saccharum spp. hybrid clone C91-301, Salsola lanata, Salsola laricifolia,Salsola grandis, Salsola tragus Sorghum bicolor, Synechocystis PCC 6803, Phaeodactylum tricornutum (strain CCAP 1055/1), Pinus strobus, Thalassiosira weissfloggi, Zea mays, Zostera muelleri
predicted reactivity:	Cucumis sativus (PEPC1, PEPC2, PEPC3), Flaveria bidentis, Flaveria trinervia, Glycine max, Lupinus albus, Mammillaria thornberi, Manihot esculenta, Manihot obovata, Medicago sativa, Morinda citrifolia, Nannochloropsis gaditana CCMP526, Nopalea gaumeri, Opuntia macbridei, Pachycereus pringlei, Saccharum spp, Solanum tuberosum, Spinacia oleracea, Streptanthus tortuosus, Pachycereus hollianus, Pisum sativa, Phaseolus vulgaris, Populus sp.,Triticum aestivum, algae, diatoms: Thalassiosira pseudonana, other species: Salmonella sp., Schiedea hookeri, Shigella sp. Schiedea sarmentosa, Streptanthus farnsworthianus, Tacinga saxatilis,Yersinia sp. Vibrio sp., Quercus sp. Species of your interest not listed? Contact us
not reactive in:	Methanothermobacter thermautotrophicus
More images:	Reactant: Arabidopsis thaliana (Thale cress) Application: Western Blotting Pudmed ID: 24723409 Journal: J Exp Bot Figure Number: 4A Published Date: 2014-07-01 First Author: Pinto, H., Sharwood, R. E., et al. Impact Factor: 6.088 Open Publication Immunoblot analyses of photosynthetic enzymes. Examples of immunoblot analysis for the photosynthetic proteins Rubisco (A), PEPC (B), NADP-ME (C), and PEP-CK (D) extracted from leaves of selected grass species grown at glacial (180 ?l l–1, G) or ambient (400 ?l l–1, A) [CO2]. Reactant: Zea mays (Maize/Corn) Application: Western Blotting Pudmed ID: 26208645 Journal: J Exp Bot Figure Number: 10C Published Date: 2015-11-01 First Author: Chen, J., Wu, F. H., et al. Impact Factor: 6.088 Open Publication Western blot analysis of RuBISCO LSU (A) and PEPC (C) of maize plants. Maize seedlings were pre-treated with 100 µM NaHS for 8 d and then grown in a nutrient solution containing 1 µM Fe(III)-EDTA or 50 µM Fe(III)-EDTA for 12 d. Relative expression level is shown as the ratio of RuBISCO LSU:?-actin (B) and PEPC:?-actin (D) using Quantity One software. Data are presented as means ± SE. Columns labelled with different letters indicate significant differences at P<0.05. –Fe, 1 µM Fe; –Fe+NaHS, seedlings were pre-treated with 100 µM NaHS and then treated with 1 µM Fe; +Fe, 50 µM Fe; +Fe+NaHS, seedlings were pre-treated with 100 µM NaHS and then treated with 50 µM Fe. Reactant: Echinochloa (Barnyard grass) Application: Western Blotting Pudmed ID: 29659931 Journal: J Exp Bot Figure Number: 5A Published Date: 2018-05-25 First Author: Sonawane, B. V., Sharwood, R. E., et al. Impact Factor: 6.088 Open Publication Immunoblot analysis of photosynthetic enzymes. Immunoblot analysis for the photosynthetic proteins Rubisco, PEPC, PEP-CK, and NADP-ME extracted from leaves of eight C4 grasses belonging to three biochemical subtypes in control (C) or shade (S) environments. Loaded volumes varied between 4 ?l and 15 ?l in order to normalize the protein content to a common leaf area. Because of the small gel size, a limited number of samples (8–9) was loaded on an individual gel. Finally, all immunoblots of the studied protein and species were arranged in a composite figure. For uniform visualization, gamma settings of individual images were adjusted. A protein ladder was used for individual immunoblots; for simplicity, band size is referred to numerically. Reactant: Oryza sativa (Asian rice) Application: Western Blotting Pudmed ID: 30499169 Journal: Mol Plant Pathol Figure Number: 3A Published Date: 2019-04-01 First Author: Hui, S., Shi, Y., et al. Impact Factor: 5.418 Open Publication Expression patterns of OsImp?1a and OsImp?1b. (A) OsImp?1a and OsImp?1b are nucleocytoplasmic?localized proteins. Total protein, the nucleus?depleted fraction and nucleus?enriched fraction were loaded onto a sodium dodecylsulfate?polyacrylamide gel electrophoresis (SDS?PAGE) gel and subjected to immunoblot analysis. Histone H3 and phosphoenolpyruvate carboxylase (PEPC) were used as nuclear and cytosolic markers, respectively. T, total protein extracts; N, nucleus?enriched fraction; C, nucleus?depleted fraction. (B) Expression of OsImp?1a and OsImp?1b in different tissues. Tissues were collected at the booting stage from IR24. (C) Expression of OsImp?1a and OsImp?1b after infection with Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99.
Picture (footer):	Application example 5 µg of total protein from (1) Arabidopsis thaliana leaf extracted with Protein Extration Buffer, PEB (AS08 300), (2) Spinacia oleracea total cell, extracted with PEB, (3) Hordeum vulgare total cell extracted with PEB, (4) Zea mays total cell extracted with PEB, were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% blocking reagent (GE Healthcare) 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 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 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 the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad). 10 µg of total protein extracted freshly from Arabidopsis thaliana wt leaf tissue (At_nnon-senescent leaves), Arabidopsis thaliana wt leaf tissue (At_s senescent leaves), Pinus strobus needle tissue (PS_0,PS₃₆) with 1 M Tris-HCl, pH 6.8, 10 % SDS, 15 % sucrose, 0.5 DTT and denatured at 75°C for 5 min. were separated on 10 % Bis-Tris Nupage Novex gel (120 V/45 min. using MES buffer system) and blotted 30 min. to PVDF. Blot was blocked with 5 % non-fat milk 45 min./RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 for 1h/RT with agitation in TBS with 2 % non-fat milk or ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice for 10 min. in TBS at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:75 000 in for 1h/RT with agitation. The blot was washed as above and developed using chemiluminescent detection. Exposure time was 40 seconds. Courtesy of Dr. Christine Yao-Yun Chang and the Ensminger lab, University of Toronto, Canada

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