Quantitative Western Blot Analysis

Western blot analyses were performed as described previously (9 (link), 33 (link)). Blots were probed with specific antibodies against PBF (9 (link), 33 (link)), 1:200; HA (Covance Research Products), 1:2000; p53(D0–1) (Santa Cruz Biotechnology), 1:1000 and Rad6 (Abcam, #ab31917), 1:1000. Antigen-antibody complexes were detected using the ECL Plus chemiluminescent detection system (Amersham Biosciences). Actin expression was determined using mouse monoclonal anti-β actin antibody clone AC-15 (Sigma-Aldrich) at 1:10,000. Protein quantification was performed on cell lysates using the Bradford assay. To quantify detected bands by densitometry, blots were scanned into Photoshop (Adobe Systems) keeping all scanning parameters the same and analyzed using ImageJ software (34 ).

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Read M.L., Seed R.I., Fong J.C., Modasia B., Ryan G.A., Watkins R.J., Gagliano T., Smith V.E., Stratford A.L., Kwan P.K., Sharma N., Dixon O.M., Watkinson J.C., Boelaert K., Franklyn J.A., Turnell A.S, & McCabe C.J. (2014). The PTTG1-Binding Factor (PBF/PTTG1IP) regulates p53 activity in thyroid cells. Endocrinology, 155(4), 1222-1234.

Publication 2014

ECL Plus chemiluminescent detection system mouse monoclonal anti-β actin antibody clone AC-15

Actin Antibodies Antigen antibody complexes Assay Cell Clone Densitometry Monoclonal antibody Mouse Protein Western blot analyses

Corresponding Organization :

Other organizations : University of Birmingham, University of Ferrara, University of British Columbia, University Hospitals Birmingham NHS Foundation Trust, National Health Service

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Protocol cited in 2 other protocols

Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression of PBF
Expression of HA
Expression of p53
Expression of Rad6
Expression of β-actin

control variables

Protein quantification using the Bradford assay
Detection of antigen-antibody complexes using the ECL Plus chemiluminescent detection system
Scanning and analysis of blots using Photoshop and ImageJ software

controls

Positive control: None mentioned
Negative control: None mentioned

Annotations

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