Liver specimens taken from a total of 16 patients and HCC cells were subjected to Western blotting and proteins extracted from the specimens were resolved in 12% SDS-PAGE and electro-transferred onto PVDF membranes (350 mA for 1 h). All blots were scanned using a GeneGnome HR Bioimaging system (Syngene, Bristol, UK). Immunohistochemical staining was conducted using standard procedure detailed in Supplementary Methods , and scoring was performed independently by three investigators as previously described [41 (link)]. Primary antibodies used in this study are listed in the Supplementary Methods section of this typescript.
Genegnome hr bioimaging system
Manufactured by Syngene
Sourced in United Kingdom
The GeneGnome HR Bioimaging system is a laboratory equipment designed for high-resolution imaging of biological samples. It provides advanced imaging capabilities to support scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
3 protocols using genegnome hr bioimaging system
1
Western Blot and Immunohistochemistry of HCC
2
Western Blot Protein Analysis Protocol
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Cells were homogenized in RIPA lysis buffer (Millipore, Billerica, MA, USA) containing 1% Halt Protease and Phosphatase Inhibitor Cocktails (Thermo Scientific, Rockford, IL, USA) and incubated on ice for 30 min. After centrifugation, proteins in the supernatant were quantified using a Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of proteins were fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were then blocked with 5% non-fat milk in TBST (10 mM Tris-HCl [pH 8], 150 mM NaCl, 0.05% Tween 20) for 1 h, probed overnight with designated primary antibodies at 4 °C, then incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies. Signals were developed using Immobilon Western chemiluminescent HRP substrate (Millipore) and scanned in a GeneGnome HR Bioimaging system (Syngene, Bristol, UK). GAPDH was used as an internal control.
3
Western Blot Analysis of TP53INP1 Protein
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Proteins were extracted in radioimmunoprecipitation buffer (EMD Millipore, Billerica, MA, USA). Protein concentration was determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Samples with equal amounts of total protein were separated on 12% SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Upon blocking in TBS/Tween-20 containing 5% non-fat milk powder at room temperature for 5 min with agitation, the membranes were incubated for 1 h with anti-TP53INP1 rabbit polyclonal antibody (catalog no. AP11890b; 1:250; Abgent, Inc.) and anti-β-actin mouse monoclonal antibody (catalog no. CW0096; 1:1,000; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) at 4°C overnight. Upon incubation of the membranes with secondary antibodies (catalog no. HSP0007; 1:200; Shanghai Mjol Biological Technology Co., Ltd.) at room temperature for 3 h, immunoreactive bands were visualized by enhanced chemilumiscence using a GeneGnome HR Bioimaging System (Syngene, Frederick, MD, USA).
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