Immunoblots were performed with samples containing total protein (40 μg) and 12% NuPage Bis-Tris or 7% Tris-Acetat polyacrylamide gels (LifeTechnologies). The membranes were probed with rabbit polyclonal anti-PER1 (1:500; Abcam ab136451), rabbit polyclonal anti PER2 (1:250; ab180655), rabbit monoclonal anti-PER3 (1:2500; ab177482), rabbit polyclonal anti-DBP (1:400; ab22824), rabbit polyclonal anti-Twist1 (1:400; ab49254) and rabbit polyclonal anti-TWIST2 antibodies (1μg/ml; ab66031). The secondary antibody was a horseradish peroxidase-linked goat anti-rabbit IgG (various concentrations; ab97051), and it was detected by the chemiluminescence technique using the Immobilon Western ECL system (Millipore). For a loading control, we used a mouse monoclonal anti-p84 antibody (nuclear matrix protein 84; 1:2000; Abcam ab487). The secondary antibody was a horseradish peroxidase-linked goat anti-mouse IgG (1:5000; Abcam; ab20043). Densitometric analysis was performed using the ImageJ software and the intensity of the control lanes were set as 1.
Ab177482
Manufactured by Abcam
Sourced in United Kingdom
Ab177482 is a primary antibody produced in rabbit. It is designed for use in immunohistochemistry and western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon ecl western system, by Merck Group (1 mentions)
Ab136451, by Abcam (1 mentions)
Ab49254, by Abcam (1 mentions)
Ab180655, by Abcam (1 mentions)
Ab66031, by Abcam (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by Affinity Biosciences (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
2 protocols using ab177482
1
Immunoblotting analysis of circadian proteins
2
Western Blot Analysis of PER3 and MEK/ERK Pathway in Breast Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Breast cancer tissues or cell lines were lysed on the ice for 30 minutes and total proteins were collected using radioimmunoprecipitation (RIPA) buffer (Solarbio, China) containing phosphatase and protease inhibitors. The BCA Protein Assay Kit (Solarbio) was used for measurement of protein concentration. After heat denaturation, 30 μg of protein samples were loaded and then electrophoretically separated by 8% to 12% SDS-PAGE gel. The proteins were electrotransfered onto PVDF membranes (Millipore, USA), followed by blocking with 0.5% BSA (Solarbio) for 60 minutes. Next, the membranes were incubated with primary antibodies against PER3 (1:1000, ab177482, abcam, UK), p-ERK5 for 1 hour at room temperature. For further analysis of MEK/ERK signaling pathway, breast cancer cells were additionally treated with 50 µM of its inhibitor (PD98059, APExBIO) and activator (TPA, APExBIO, USA) for 24 hours. Finally, the protein signals were visualized by enhanced chemiluminescence reagent (Affinity Biosciences, USA).
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