Western Blot Protein Analysis

Cells were lysed in lysis buffer [44 (link)] and cell debris was removed by centrifugation at 14,000xg at 4°C for 15 min. Supernatant extracts were quantified using Bio-Rad protein assay kit. Equal amounts of protein (20-40 μg) were subjected to SDS-PAGE and transferred to PVDF membranes. After transfer, the membranes were incubated with blocking solution and sequentially blotted with primary and second antibodies. The blots were finally developed by the enhanced ECL kit (Thermo Fisher Scientific).

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Ying Y., Ueta T., Jiang S., Lin H., Wang Y., Vavvas D., Wen R., Chen Y.G, & Luo Z. (2017). Metformin inhibits ALK1-mediated angiogenesis via activation of AMPK. Oncotarget, 8(20), 32794-32806.

Publication 2017

protein assay kit enhanced ECL kit

Antibodies Assay Buffer Cell Centrifugation Membranes Protein Pvdf Rad protein Sds page

Corresponding Organization : Windsor University School of Medicine

Other organizations : Massachusetts Eye and Ear Infirmary, Harvard University, University of Miami, Tsinghua University

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Variable analysis

independent variables

Lysis buffer used to lyse cells

dependent variables

Protein expression levels quantified by SDS-PAGE and Western blotting

control variables

Centrifugation at 14,000xg at 4°C for 15 min to remove cell debris
Protein quantification using Bio-Rad protein assay kit
Equal amounts of protein (20-40 μg) loaded for SDS-PAGE
Transfer of proteins to PVDF membranes
Incubation with blocking solution and primary/secondary antibodies
Development of blots using enhanced ECL kit

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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