To assess phosphorylated extracellular signal–regulated kinase (phospho-ERK), 5 × 105 bmEos were incubated for 10 minutes at 37 °C. Increasing doses of mCCL11 were added to the wells for 30, 120 and 300 seconds. Paraformaldehyde (PFA, 1% final) was added to each well for 10 minutes at 37 °C. Cells were resuspended in PBS, and ice-cold methanol (90% final concentration) was slowly added. Cells were permeabilized overnight at −20 °C and then washed and stained with anti–phospho-ERK1/2 Alexa Fluor 647 antibody (1:500, #13148, Cell Signaling Technologies (CST)) for 60 minutes at room temperature (RT). To assess F-actin, bmEos were plated at 100,000 cells/well and incubated for 10 minutes at 37 °C. Cells were stimulated with 2, 20 or 200 ng/mL mCCL11 for 10, 20, 30 or 60 seconds. After treatment, the cells were incubated with PFA and permeabilized with PBS with 1% BSA and 0.05% TritonX-100. Cells were incubated for 20 minutes at RT in PBS with 1% BSA containing Phalloidin-Alexa Fluor 488 (Molecular Probes) at a 1:1000.
Anti phospho erk1 2 alexa fluor 647 antibody
Manufactured by Cell Signaling Technology
The Anti–phospho-ERK1/2 Alexa Fluor 647 antibody is a fluorescently-labeled antibody that specifically recognizes the phosphorylated forms of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) proteins. The Alexa Fluor 647 dye provides a far-red fluorescent signal that can be detected using appropriate instrumentation.
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2 protocols using anti phospho erk1 2 alexa fluor 647 antibody
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Kinase and Cytoskeleton Dynamics Assay
2
Kinase and Cytoskeleton Dynamics Assay
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To assess phosphorylated extracellular signal–regulated kinase (phospho-ERK), 5 × 105 bmEos were incubated for 10 minutes at 37 °C. Increasing doses of mCCL11 were added to the wells for 30, 120 and 300 seconds. Paraformaldehyde (PFA, 1% final) was added to each well for 10 minutes at 37 °C. Cells were resuspended in PBS, and ice-cold methanol (90% final concentration) was slowly added. Cells were permeabilized overnight at −20 °C and then washed and stained with anti–phospho-ERK1/2 Alexa Fluor 647 antibody (1:500, #13148, Cell Signaling Technologies (CST)) for 60 minutes at room temperature (RT). To assess F-actin, bmEos were plated at 100,000 cells/well and incubated for 10 minutes at 37 °C. Cells were stimulated with 2, 20 or 200 ng/mL mCCL11 for 10, 20, 30 or 60 seconds. After treatment, the cells were incubated with PFA and permeabilized with PBS with 1% BSA and 0.05% TritonX-100. Cells were incubated for 20 minutes at RT in PBS with 1% BSA containing Phalloidin-Alexa Fluor 488 (Molecular Probes) at a 1:1000.
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