Immunoblot Analysis Protocol

Antibodies used for immunoblots are listed in Supplementary Table 10. Membranes were incubated with blocking buffer (5% milk/TBST) with primary antibody overnight at 4 °C followed by probing the membranes with HRP-conjugated secondary antibodies for 1 h at room temperature. Lastly, the signals were visualized using ECL reagent (Thermo Scientific) and imaged in Azure C300 gel imaging system (Azure Biosystems). Immunoblot data were quantified using NIH Image J software for Windows [30 (link)].

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Rasha F., Boligala G.P., Yang M.V., Martinez-Marin D., Castro-Piedras I., Furr K., Snitman A., Khan S.Y., Brandi L., Castro M., Khan H., Jahan N., Almodovar S., Melkus M.W., Pruitt K, & Layeequr Rahman R. (2023). Dishevelled 2 regulates cancer cell proliferation and T cell mediated immunity in HER2-positive breast cancer. BMC Cancer, 23, 172.

Publication 2023

ECL reagent Azure C300 gel imaging system

Antibodies Antibody Azure Buffer Immunoblot Membranes Milk

Corresponding Organization :

Other organizations : Texas Tech University Health Sciences Center, Texas Tech University

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Variable analysis

independent variables

Type of primary antibody used for immunoblot

dependent variables

Protein expression levels as measured by immunoblot signal intensity

control variables

Blocking buffer (5% milk/TBST)
Incubation time and temperature for primary antibody (overnight at 4 °C)
Incubation time and temperature for secondary antibody (1 h at room temperature)
Visualization method (ECL reagent and Azure C300 gel imaging system)

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