Leaf disks from 4- or 5-week-old plants were incubated in the ddH2O overnight at 23 °C, and then treated with 200 μM ATPγS, 1 μM flg22, or 100 μg/ml chitin for 0, 15, 30, or 60 min. Total protein was extracted with extraction buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% Triton-X 100, and 1x protease inhibitor for 30 min on ice. The extracted total proteins were separated by 10% (w/v) SDS-PAGE gel and detected by immunoblotting with anti-phospho-p44/p42 MAPK antibody (Cell Signaling, 9101, dilution, 1:1000).
Anti phospho p44 p42 mapk antibody
Manufactured by Cell Signaling Technology
The Anti-phospho-p44/p42 MAPK antibody is a laboratory reagent used to detect and quantify the levels of phosphorylated forms of the p44/p42 mitogen-activated protein kinases (MAPK). This antibody specifically recognizes the dually phosphorylated, active forms of these kinases.
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Lab products found in correlation
3 protocols using anti phospho p44 p42 mapk antibody
1
MAPK Activation in Plant Immunity
2
ROS Production and MAPK Assay
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ROS production was determined enzymatically with reaction buffer in 96-well plates containing 20 μg/ml Horseradish peroxidase (HRP), 20 μM L-012 (Waco), and either 1 μM purified protein or ddH2O (mock). Then luminescence detection was performed using a microwell plate reader. Light emissions were measured at 3-minute intervals [82 (link)–84 (link)]. For the MAPK assay, leaves treated with 1 μM protein were extracted at 30 minutes post-treatment, then MAPK was detected with anti-phospho-p44/p42 MAPK antibody (Cell Signaling).
3
Protein Extraction and MAPK Activation
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Ten seedlings were immersed in sterile water overnight. Peptides were then added to a final concentration of 1 µM for 5–15 minutes induction. After induction, the seedlings were snap-frozen in liquid nitrogen and ground to a fine powder, from which total protein was extracted by suspension in 50 mM HEPES (pH 6.8), 150 mM NaCl, 1% (w/v) SDS, 2 mM DTT, 10 mM NaF, 10 mM NaVO3, 5 mM EDTA, 1× protease inhibitor cocktail (Roche). An anti-phospho p44/p42 MAPK antibody (Cell Signaling Technology) was used to detect active MPK6 and MPK3 via immunoblotting.
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