Total protein from cells and tissues was extracted with radioimmunoprecipitation assay buffer, and total protein content was determined with the BCA protein assay (Sigma-Aldrich, St. Louis, MO) with BSA as a standard. Proteins were separated by SDS-PAGE with Bis-Tris gels (Bio-Rad Laboratories), transferred onto polyvinylidene fluoride membranes at 100 V constant for 1 h, and incubated with an appropriate primary antibody against phosphorylated (p)AktThr308 (13038; Cell Signaling Technology), pAktSer473 (4060; Cell Signaling Technology), total Akt (4691; Cell Signaling Technology), p-p44/42MAPKThr202/Tyr204 (9101; Cell Signaling Technology), and total p44/42 MAPK (9107; Cell Signaling Technology) IGF-1R (9750; Cell Signaling Technology) or InsRβ (9H4; Santa Cruz Biotechnology) overnight at 4°C (24 (link),31 (link)). Following a 1-h incubation with secondary antibody, Clarity Western ECL Substrate (Bio-Rad Laboratories) was applied to the membrane and bands were visualized with a ChemiDoc bio-imager (Bio-Rad Laboratories) to first pixel saturation and densitometry performed with Image Lab (Bio-Rad Laboratories).
P p44 42mapkthr202 tyr204
Manufactured by Cell Signaling Technology
The P-p44/42MAPK(Thr202/Tyr204) product is an antibody that detects phosphorylation of p44/42 MAPK (Erk1/2) at Thr202 and Tyr204. It is used to monitor the activation state of the p44/42 MAPK signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
P akt ser473, by Cell Signaling Technology (2 mentions)
Total p44 42 mapk, by Cell Signaling Technology (2 mentions)
Chemidoc bio imager, by Bio-Rad (2 mentions)
Bca protein assay, by Merck Group (2 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
Bis tris gel, by Bio-Rad (1 mentions)
Phosphorylated p aktthr308, by Cell Signaling Technology (1 mentions)
Igf 1r, by Cell Signaling Technology (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Total s6, by Cell Signaling Technology (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
2 protocols using p p44 42mapkthr202 tyr204
1
Western Blot Analysis for Phosphorylated Proteins
2
Western Blotting for Protein Expression Analysis
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Western blotting was performed as described (37 (link)). Protein was extracted from frozen tissues in RIPA buffer and total protein content was determined using the BCA protein assay (Sigma, St. Louis, Mo) with BSA as a standard. Proteins were separated by SDS-PAGE, transferred onto PVDF membranes and incubated with an appropriate primary and secondary antibody. Equal loading and transfer was confirmed by staining, imaging and quantifying the original gel, using Biorad stain-free gel technology. Immunoblotting was performed for pS6 (#5364), total S6 (#2217), pAkt Ser473 (#4060), total Akt (#4691), p-p44/42MAPKThr202/Tyr204 (#9101) total p44/42 MAPK (#4695), and β-catenin (#8480), all from Cell Signaling. Bands were visualized by chemiluminescence to first indication of pixel saturation using a Biorad Chemidoc bioimager and densitometry performed using Image Lab 4.1 (Biorad, Hercules, CA).
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