Immunoprecipitation and Western Blot Analysis

Total protein was extracted from HCT-116 cells and protein concentration was measured using a BCA Protein Assay Kit (KeyGen). Total protein lysate (2 mg) was used for each immunoprecipitation (IP) using specific antibody or the corresponding IgG control. Protein A agarose beads (GE Healthcare, Uppsala, Sweden) were added to the cells and then were incubated overnight at 4 °C. Washed precipitated proteins were analyzed by western blotting. The IP, western blotting and GST pull-down assays were used in this study as described previously in detail.^{40 (link)}

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Zhang X., Zhang X., Li Y., Shao Y., Xiao J., Zhu G, & Li F. (2017). PAK4 regulates G6PD activity by p53 degradation involving colon cancer cell growth. Cell Death & Disease, 8(5), e2820-.

Publication 2017

BCA Protein Assay Kit Protein A agarose beads

Agarose Antibody Assays Cells Hct 116 cells Immunoprecipitation Protein a Proteins Pull

Corresponding Organization :

Other organizations : China Medical University, Jinzhou Medical University

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Variable analysis

independent variables

Antibody used for immunoprecipitation (IP)

dependent variables

Protein expression and interactions detected by western blotting

control variables

Protein concentration (2 mg total protein lysate used for each IP)
Incubation time for IP (overnight at 4 °C)
Protein A agarose beads used for IP
IgG control for IP

controls

Positive control: Not specified
Negative control: IgG control for IP

Annotations

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