In-vivo ubiquitinated Nrf2 levels were estimated as described earlier.49 (link) To minimize non-specific precipitation, cell lysates were incubated with normal goat serum for 1 h before precipitation with Nrf2 antibody (developed in goat). For western blot transfer, BioTrace PVDF (polyvinylidene fluoride) membranes (Pall German Laboratory, MI, USA, USA) were used and visualized using the Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate Kit (Millipore Corporation, Billerica, MA, USA) on ImageQuant LAS 500 detection system (GE Healthcare, Upsala, Sweden). All western blotting images are representative of three independent experiments. The bands from western blotting were quantified by the ImageJ 1.47v software (National Institutes of Health, Bethesda, MD, USA).
Imagequant las 500 detection system
Manufactured by GE Healthcare
Sourced in United States
The ImageQuant LAS 500 detection system is a digital imaging device designed for the analysis of blots and gels. It captures high-quality images of chemiluminescent, fluorescent, and colorimetric samples. The system utilizes a charge-coupled device (CCD) camera and advanced optics to provide reliable and sensitive detection of a variety of sample types.
Automatically generated - may contain errors
2 protocols using imagequant las 500 detection system
1
Quantification of Ubiquitinated Nrf2 In-vivo
2
Western Blot Analysis of Notch2 Expression
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Cells were lysed in 1X lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein lysates were separated by SDS-PAGE and transferred onto nitrocellulose membrane (Amersham, GE Healthcare Life Sciences, Inc.). The membranes were then incubated with specific primary antibodies Notch2 (Cell Signaling Technology, USA) and GAPDH (Bio-Rad, USA) at 4°C overnight. This was followed by incubation with HRP-conjugated secondary antibody (Bio-Rad, USA). Immunoblotting signals were visualized using the ImageQuant™ LAS 500 detection system (GE Healthcare Life Sciences, Inc., Chicago, IL, USA) [44 (link)].
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