Total protein of cell lines was isolated using a total protein extraction kit (Boster Biological Technology, Wuhan, China); concentration of protein was determined using a bicinchoninic acid kit (Pierce; Thermo Fisher Scientific, Inc.) at 570 nm absorption value. Western blotting was conducted as described by Li et al. [9 (link)]. Briefly, lysate protein was separated using SDS-PAGE, using 10% gel, and was transferred to nitrocellulose membrane. The expression signal was detected using an enhanced chemiluminescence system (EMD Millipore, Billerica, MA, USA). Primary antibodies used in this assay were as follow: iASPP primary antibody (ab115605, Abcam, UK), GAPDH primary antibody (ab9385, Abcam, UK), and anti-rabbit secondary antibody (ab191866, Abcam, UK).
Protocol detail
Protein Isolation and Western Blotting
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Anti antibody
Antibodies
Antibody
Assay
Bicinchoninic acid
Biological
Cell lines
Chemiluminescence
Gapdh
Membrane
Nitrocellulose
Protein
Rabbit
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Corresponding Organization :
Other organizations : Shandong University, Shandong Tumor Hospital, Shandong First Medical University, Yantaishan Hospital
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Variable analysis
independent variables
- None explicitly mentioned
- Expression signal of iASPP protein
- GAPDH protein expression (used as a loading control)
- Positive control: None mentioned
- Negative control: None mentioned
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