Nodules (4 g) were ground into powder in liquid nitrogen, and protein was extracted by 5 ml extraction buffer (50 mM Tris-MES [morpholineethanesulfonic acid], pH 8, 0.5 M sucrose, 1 mM MgCl2, 10 mM EDTA, 5 mM dithiothreitol [DTT], and protease inhibitor cocktail). The extract was centrifuged at 10,000 × g at 4°C for 30 min. Protein concentration was determined by using the Bradford method (A53225; ThermoFisher). Then, 2 μg protein was separated by SDS-PAGE (15% acrylamide gel) and transferred to nitrocellulose filter membranes by wet electrotransfer (Bio-Rad). The membrane was blocked in 1× Tris-buffered saline (TBS) buffer containing 5% nonfat milk. The blocked membrane was further incubated with anti-phospho-p44/p42 MAPK (anti-pTEpY; Cell Signaling Technology) diluted at 1:2,000 overnight at 4°C and subsequently with secondary antibody diluted at 1:5,000 for 1 h. Finally, the bands were detected using chemiluminescent horseradish peroxidase (HRP) substrate, and the image was obtained by using Tanon 5200. Three independent biological replicates were used.
Anti phospho p44 p42 mapk anti ptepy
Manufactured by Cell Signaling Technology
Sourced in United States
Anti–Phospho-p44/p42 MAPK (anti-pTEpY) is a lab equipment product that detects the phosphorylated form of p44/p42 mitogen-activated protein kinases (MAPK). It binds to the phosphorylated threonine and tyrosine residues in the activation loop of these MAPKs.
Automatically generated - may contain errors
Lab products found in correlation
Semi dry electrotransfer, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Quick start bradford dye reagent, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp substrate, by Merck Group (1 mentions)
Protease inhibitor cocktail s8830, by Merck Group (1 mentions)
Chemiluminescent hrp substrate, by Merck Group (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Coomassie blue, by Merck Group (1 mentions)
As09 491, by Agrisera (1 mentions)
As09 484, by Agrisera (1 mentions)
As07 266, by Agrisera (1 mentions)
As07 260, by Agrisera (1 mentions)
5 protocols using anti phospho p44 p42 mapk anti ptepy
1
Phospho-p44/p42 MAPK Immunoblotting
2
Freezing Stress Response of B. rapa
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B. rapa seedlings were grown on 1/2 MS medium, containing 0.8% agar, at 22°C (16 h light/8 h) for 14 days and exposed to the freezing treatment. The freezing treatment protocol is similar to those in the plant materials and growth conditions section mentioned above. Materials were collected from the NA treatment and CA treatment groups (pretreated at 4°C for 72 h) at −4°C (3 h) and −8°C (3 h) to extract total proteins with a protein extraction buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 1 mM EDTA, 20 mM NaF, and 1× protease inhibitor cocktail). The protein concentration of each sample was adjusted to the same level using Quick Start Bradford Dye Reagent (Bio-Rad, California, United States). Total proteins were separated via 10% SDS/PAGE and transferred onto PVDF membranes (Bio-Rad). Immunoblotting was performed using anti–Phospho-p44/p42 MAPK (anti-pTEpY) (1:2,000, Cell Signaling Technology, Danvers, United States).
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B. rapa seedlings were grown on 1/2 MS medium, containing 0.8% agar, at 22°C (16 h light/8 h) for 14 days and exposed to the freezing treatment. The freezing treatment protocol is similar to those in the plant materials and growth conditions section mentioned above. Materials were collected from the NA treatment and CA treatment groups (pretreated at 4°C for 72 h) at −4°C (3 h) and −8°C (3 h) to extract total proteins with a protein extraction buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 1 mM EDTA, 20 mM NaF, and 1× protease inhibitor cocktail). The protein concentration of each sample was adjusted to the same level using Quick Start Bradford Dye Reagent (Bio-Rad, California, United States). Total proteins were separated via 10% SDS/PAGE and transferred onto PVDF membranes (Bio-Rad). Immunoblotting was performed using anti–Phospho-p44/p42 MAPK (anti-pTEpY) (1:2,000, Cell Signaling Technology, Danvers, United States).
3
Soybean Protein Extraction and Western Blotting
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Protein was extracted from flg22-treated soybean leaf tissues in an extraction buffer (50 mm Tris-MES, pH 8, 0.5 m Suc, 1 mm MgCl2, 10 mm EDTA, and 5 mm DTT as described [49 (link)]. For Western blotting, proteins were separated by SDS-PAGE (10% acrylamide gel) and transferred to PVDF membranes (Millipore, Burlington, MA, USA) by semidry electrotransfer (Bio-Rad) followed by incubation with the anti-phospho-p44/p42 MAPK (anti-pTEpY) diluted at 1:2000 (Cell Signaling Technology, Danvers, MA, USA). The bands were visualized using horseradish peroxidase (HRP) substrate (Millipore). Coomassie Blue–stained gel (CBS) was used as an equal loading control.
4
Soybean Leaf Protein Extraction and Analysis
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Protein was extracted from soybean leaf tissues using extraction buffer (50 mM Tris-MES pH 8.0, 0.5 M sucrose, 1 mM MgCl2, 10 mM EDTA, 5 mM DTT) with protease inhibitor cocktail S8830 (Sigma-Aldrich, St. Louise, MO, USA) added as described by [23 (link)]. The extract was centrifuged at 12,000 rpm at 4 °C for 30 min and the supernatant was collected. For immunoblotting, the extracted proteins were separated by SDS-PAGE (10% acrylamide gel) and transferred to PVDF membrane (Millipore, Billerica, MA, USA) by semi-dry electro-transfer (Bio-Rad, Hercules, CA, USA). The membrane was blocked in 1× TBST buffer containing 5% milk powder for 2 h. After washing, the membrane was further incubated with anti–Phospho-p44/p42 MAPK (anti-pTEpY) diluted at 1:3000 (Cell Signaling Technology, Danvers, MA, USA), followed by incubation with secondary antibody diluted at 1:7500. The bands were visualized by incubating with chemiluminescent HRP substrate (Millipore, Billerica, MA, USA).
5
Western Blot Analysis of Plant Proteins
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Proteins were separated by SDS-PAGE (10% acrylamide gel) using the technique from Schägger and von Jagow [92 (link)]. Equal amounts of total protein were used in all protein analyses performed, the same loading amount was verified running parallel gels used for blotting and Coomassie Blue (Sigma-Aldrich Corp. St. Louis, MO) staining. The gel was transferred onto PVDF membranes (Immobilon-P, Millipore Corp. Bedford, MA) by electro-transfer at 22 V for 2.5 h The membrane was enhanced with Western Blot Signal Enhancer (Pierce®, Thermo Scientific, IL, USA) and blocked in TBST buffer containing 2% free fat milk powder and further incubated with primary antibody and second antibody. Finally, the bands were detected using alkaline phosphatase reaction (1:2500, Millipore). Antibodies used for immunoblotting were as follows: anti PIP2;1, PIP2;2, PIP2;3 (1:1000, Agrisera AS09 491), anti-Na+/H+ exchanger 1 (1:1000, Agrisera AS09 484), anti-SMT1 (1:1000, Agrisera AS07 266), anti-H+-ATPase (1:10,000, Agrisera AS07 260), anti-14-3-3 proteins (1:1000, Agrisera), and anti-Phospho-p44/p42 MAPK (anti-pTEpY) (1:2000, Cell Signaling Technology).
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