Cultures were grown on chamber slides and fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed with PBS then stained using standard protocols. Briefly, the fixed cells were permeabilized with 0.1% Triton X-100 then blocked using 10% serum. The primary antibodies used were anti-Mapk1 (1:800), anti-phospho-Neu (1:50)(Santa Cruz Biotech), anti-Notch2 (1:200)(Cell Signaling Technologies); each was applied and incubated overnight at 4°C. Secondary antibodies were conjugated to Alexa 488 or Alexa 568 (1:200)(Life Technologies) and incubated for 60 min at room temperature. Slides were coverslipped with ProLong Antidfade mounting media containing DAPI (Life Technologies). Images were collected using a Leica TCS SPE confocal microscope at the same laser and acquisition setting. To ensure the laser power and imaging settings were not acquiring autofluoresence, a comparison image was generated from using an unstained sample. Images were analyzed using Image J and Adobe Photoshop.
Anti mapk1
Manufactured by Santa Cruz Biotechnology
Anti-Mapk1 is a laboratory reagent used in scientific research. It functions as an antibody that specifically binds to the Mapk1 protein, also known as Erk2. Mapk1 is a member of the mitogen-activated protein kinase (MAPK) family and plays a key role in cellular signal transduction pathways.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Alexa 568, by Thermo Fisher Scientific (1 mentions)
Tcs spe confocal microscope, by Leica (1 mentions)
Anti notch2, by Cell Signaling Technology (1 mentions)
Ecl kit, by Santa Cruz Biotechnology (1 mentions)
Sc 81178, by Santa Cruz Biotechnology (1 mentions)
Chemiimager 4000, by Bio-Techne (1 mentions)
Anti lc3, by Santa Cruz Biotechnology (1 mentions)
2 protocols using anti mapk1
1
Immunofluorescence Staining and Imaging
2
Quantifying MAPK1 and LC3 Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Primary antibodies used in these experiments were anti-MAPK1 (1:1000, Santa Cruz
Biotechnology), anti-LC3 (1: 1000, Santa Cruz Biotechnology), and β-actin protein (1:
1000; sc-81178; Santa Cruz Biotechnology). Cells from each group were digested (50 mM
Tris–HCl (pH 6.8), 10 mM ethylenediaminetetraacetic acid (EDTA), 2% sodium dodecyl sulfate
(SDS), 5 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride) and then the
supernatants were collected after centrifugation of 15,000 × g for 1 h.
Cell lysate (100 ml) was collected to perform protein quantification using the Bradford
method. Equal amounts of protein (20 mg) from each cell sample were separated by SDS-
polyacrylamide gel electrophoresis (PAGE) on an 8% polyacrylamide gel and transferred to
polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA, USA). This membrane was
incubated overnight with a primary antibody (dilution 1:1000) at 41°C, then incubated with
a horse radish peroxidase-conjugated secondary antibody (Zymed Laboratory, San Francisco,
CA, USA) for 1 h at room temperature. Detection of reactive antigens was performed using
an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology). The resulting image
was analyzed with ChemiImager 4000 (Alpha Innotech, San Leandro, CA, USA) for protein band
densitometry.
Biotechnology), anti-LC3 (1: 1000, Santa Cruz Biotechnology), and β-actin protein (1:
1000; sc-81178; Santa Cruz Biotechnology). Cells from each group were digested (50 mM
Tris–HCl (pH 6.8), 10 mM ethylenediaminetetraacetic acid (EDTA), 2% sodium dodecyl sulfate
(SDS), 5 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride) and then the
supernatants were collected after centrifugation of 15,000 × g for 1 h.
Cell lysate (100 ml) was collected to perform protein quantification using the Bradford
method. Equal amounts of protein (20 mg) from each cell sample were separated by SDS-
polyacrylamide gel electrophoresis (PAGE) on an 8% polyacrylamide gel and transferred to
polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA, USA). This membrane was
incubated overnight with a primary antibody (dilution 1:1000) at 41°C, then incubated with
a horse radish peroxidase-conjugated secondary antibody (Zymed Laboratory, San Francisco,
CA, USA) for 1 h at room temperature. Detection of reactive antigens was performed using
an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology). The resulting image
was analyzed with ChemiImager 4000 (Alpha Innotech, San Leandro, CA, USA) for protein band
densitometry.
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