Total RNA from liver tissue was isolated using Trizol reagent (Life Technologies, Carlsbad, CA) purified with the Qiagen RNeasy mini kit and treated with DNase I in order to remove any trace of genomic DNA using RNase-Free DNase Set (Qiagen, Valencia, CA) according to the manufacturer’s protocol. RNA concentrations and purity were determined by u.v. spectrophotometry (A260/280>1.8 and A260/240>1.7) and integrity was verified by the intensities of 28S and 18S rRNA bands on a denaturing agarose gel visualized on a FluorChem IS-8800 Imager (Alpha Innotech, San Leandro, CA).
Fluorchem is 8800 imager
Manufactured by Bio-Techne
The FluorChem IS-8800 Imager is a compact and versatile fluorescence imaging system designed for a wide range of applications. It utilizes high-resolution digital imaging technology to capture and analyze fluorescent signals from various samples.
Automatically generated - may contain errors
3 protocols using fluorchem is 8800 imager
1
Total RNA Isolation from Liver Tissue
2
Quantification of Protein Isoforms by Western Blot
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Whole cell lysate protein (30 (link)) was assayed for individual CYP isoforms and transcription factors by Western blotting (31 (link)). Briefly, 50 µg of protein were electrophoresed on 1.00 mm-thick SDS-polyacrylamide (10–12%) gels and electroblotted onto nitrocellulose membranes. The blots were probed with monoclonal anti-rat CYP2C11 and anti-rat CYP3A2 (Detroit R & D Inc., Detroit, MI), monoclonal anti-rat CYP2C6 and polyclonal anti-SOCS2 (Santa Cruz Biotechnology, Inc.), monoclonal anti-pSTAT5 and polyclonal anti-pJAK2 (Cell Signaling Technology) and polyclonal anti-JAK2 (Upstate Cell Signaling Solutions) and detected with an enhanced chemiluminescence kit (Amersham, GE Healthcare Bio-Sciences Corp). Signals were normalized to a known control sample, for example, pSTAT5, pJAK2, etc., repeatedly run on all blots and/or to the expression of β-actin (Sigma–Aldrich). The protein signals were scanned and the densitometric units were obtained as integrated density values quantitated using a FluorChem IS-8800 Imager (Alpha Innotech) software supplied with the gel documentation system.
3
Isolation and Characterization of Total Liver RNA
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Total RNA from liver tissue was isolated using Trizol reagent (Life Technologies), purified with Qiagen RNeasy mini kit, and treated with DNase I in order to remove any traces of genomic DNA using RNase-Free DNase Set (Qiagen) according to the manufacturer’s protocol. RNA concentrations and purity were determined by UV spectrophotometry (A260/280 > 1.8 and A260/240 > 1.7) and integrity was verified by the intensities of 28S and 18S rRNA bands on a denaturing agarose gel visualized on a FluorChem IS-8800 Imager (Alpha Innotech, San Leandro, CA).
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