Cells were washed with phosphate-buffered saline and harvested in 100 μl mammalian protein extraction reagent lysis buffer. Samples were homogenized by passing through 26-gauge needles for 10 times and centrifuged at 13,000 rpm for 5minutes at 4°C to prepare the supernatant. The protein concentration was determined by BCA protein assay (Thermo Scientific, Rockford, IL). Total protein (10 μg) was subjected to 4%–12% Bis-Tris Novex Gel electrophoresis and electroblotted onto nitrocellulose membranes. The membranes were probed sequentially with anti-p-Stat3 (Cell Signaling Technology (CST), Danvers, MA), anti-Stat3 (CST), anti-p-Akt (CST), anti-Akt (CST), p-Erk1/2 (Promega, Madison, WI), Erk1/2 (CST, #9102), and anti-GAPDH (Santa cruz Biotechnology Inc. Santa Cruz, CA). Blots were incubated with horseradish peroxidase-coupled anti-rabbit IgG or anti-mouse IgG and developed with ECL plus (GE Healthcare). Membranes were stripped between probing by incubation for 15minutes at room temperature with 1X ReBlot Plus Strong Antibody Stripping Solution (Millipore, Billerica, MA). Chemiluminescence was recorded with a Fuji LAS-3000 luminescent image analyzer.
P erk1 2
Manufactured by Promega
Sourced in Spain
P-Erk1/2 is a laboratory product that enables the detection and quantification of phosphorylated forms of the extracellular signal-regulated kinases 1 and 2 (ERK1/2). It provides a means to assess the activation status of this important signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti p stat3, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Las 3000 luminescent image analyzer, by Fujifilm (1 mentions)
Reblot plus strong antibody stripping solution, by Merck Group (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase, by Bio-Rad (1 mentions)
Bcl2 associated x (bax), by Promega (1 mentions)
α tubulin, by Merck Group (1 mentions)
P jnk, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Santa Cruz Biotechnology (1 mentions)
2 protocols using p erk1 2
1
Western Blot Analysis of Signaling Proteins
2
Protein Extraction and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Total protein extracts were obtained by adding the lysis buffer: 25 mM HEPES pH 7.5, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 20 mM β-glicerophosphate, 0.1 mM Na3VO4, 0.1% triton X-100; in the presence of protease inhibitors78 (link). Twenty μg of protein per sample were loaded and resolved using 8%, 10% or 15% SDS-PAGE polyacrylamide gels, and then transferred onto PVDF membranes, followed by immunodetection using appropriate antibodies, purchased from Santa Cruz Technology: Mcl-1 (sc-819), Cell Signaling Technology: BIM, BID, BAX (#9942), p-p38 (1:2000, #4631), p-p53ser15 (#9284), p-ERK1/2 (1:2000, #9106), p-CHK1ser345 and p-CHK2ser19 (#9931), Promega Corporation-Spain: p-JNK (#V7932), or Sigma-Aldrich: α-tubulin (1:10000, #TP026). Unless indicated, all antibodies were diluted 1:1000. Secondary antibodies conjugated with horseradish peroxidase were purchased from BioRad, and chemiluminescence detection was performed using ECL (Santa Cruz Biotechnology).
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