Comprehensive Western Blot Assay Protocol

Western blotting was performed exactly as described^{27 (link)}. The primary antibodies used were: anti-β-actin (Sigma, Deisenhofen, Germany; A5316), anti-MDM2 (Santa Cruz, Heidelberg, Germany (sc-965)), anti-p21^Cip/Waf (Santa Cruz; sc-397), anti-p53 (Santa Cruz; sc-126), and anti-GAPDH (Sigma; G9545). Secondary antibody F(ab′)2 fragments coupled to horseradish peroxidase and specific for rabbit-IgG (No. 111-036-045) or mouse-IgG (No. 115-036-072) were obtained from Jackson ImmunoResearch, Newmarket, UK. A freshly made solution of luminol (2.5 mM), p-coumaric acid (0.2 mM) and H₂O₂ (0.01%) in 100 mM Tris-HCl (pH 8.8) was used as reagent for chemiluminescent detection.

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Munawar U., Roth M., Barrio S., Wajant H., Siegmund D., Bargou R.C., Kortüm K.M, & Stühmer T. (2019). Assessment of TP53 lesions for p53 system functionality and drug resistance in multiple myeloma using an isogenic cell line model. Scientific Reports, 9, 18062.

Publication 2019

anti-β-actin anti-MDM2 anti-p53 anti-GAPDH F(ab′)2 fragments

Actin Antibodies Antibody fragments Gapdh H2o2 Horseradish peroxidase Luminol Mdm2 Mouse P coumaric acid Rabbit Tris

Corresponding Organization :

Other organizations : Comprehensive Cancer Center Mainfranken, Research Institute Hospital 12 de Octubre, Universitätsklinikum Würzburg

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Variable analysis

independent variables

Primary antibodies used: anti-β-actin, anti-MDM2, anti-p21Cip/Waf, anti-p53, and anti-GAPDH

dependent variables

Chemiluminescent detection of protein levels

control variables

Western blotting protocol was performed exactly as described in the referenced publication (27)
Secondary antibody F(ab′)2 fragments coupled to horseradish peroxidase and specific for rabbit-IgG or mouse-IgG were used
Reagents for chemiluminescent detection: freshly made solution of luminol (2.5 mM), p-coumaric acid (0.2 mM) and H2O2 (0.01%) in 100 mM Tris-HCl (pH 8.8)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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