Western Blotting of Microsomal P450s

Microsomal fractions were prepared as described previously [25 (link)] and stored at − 70°C until required. Microsomal protein concentrations were determined using the Bio-Rad Protein Assay Reagent (Bio-Rad Laboratories) and Western blot analysis was preformed as described previously [26 (link)] using polyclonal antisera to rat P450s and POR [27 (link),28 (link)]. Immunoreactive proteins were detected using horseradish peroxidase (HRP)-conjugated polyclonal goat anti-rabbit, anti-mouse or anti-sheep immunoglobulin secondary antibodies, as appropriate (Dako). Bands were visualized using Immobilon™ chemiluminescent HRP-conjugated substrate (Millipore) and a FUJIFILM LAS-3000 mini imaging system (Fujifilm UK.). Densitometric analysis was performed using Multi Gauge V2.2 software (Fujifilm UK).

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Henderson C.J., McLaughlin L.A., Osuna-Cabello M., Taylor M., Gilbert I., McLaren A.W, & Wolf C.R. (2015). Application of a novel regulatable Cre recombinase system to define the role of liver and gut metabolism in drug oral bioavailability. The Biochemical journal, 465(3), 479-488.

Publication 2015

Bio-Rad Protein Assay Reagent horseradish peroxidase (HRP)-conjugated polyclonal goat anti-rabbit, anti-sheep immunoglobulin secondary antibodies Immobilon™ chemiluminescent HRP-conjugated substrate LAS-3000 mini imaging system Multi Gauge V2.2 software

Anti antibodies Anti immunoglobulin Antibodies Antisera Assay Densitometric Goat Horseradish peroxidase Immobilon Immunoglobulin Microsomal Mouse Proteins Rabbit Sheep Western blot analysis

Corresponding Organization :

Other organizations : Ninewells Hospital, University of Dundee

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Microsomal protein concentrations
Immunoreactive proteins detected using HRP-conjugated secondary antibodies
Densitometric analysis of protein bands

control variables

Microsomal fractions prepared as described previously [25]
Microsomal fractions stored at − 70°C until required
Western blot analysis performed as described previously [26]
Polyclonal antisera to rat P450s and POR used [27,28]
HRP-conjugated polyclonal goat anti-rabbit, anti-mouse or anti-sheep immunoglobulin secondary antibodies used, as appropriate (Dako)
Immobilon™ chemiluminescent HRP-conjugated substrate (Millipore) used
FUJIFILM LAS-3000 mini imaging system (Fujifilm UK) used for visualization
Multi Gauge V2.2 software (Fujifilm UK) used for densitometric analysis

controls

Positive control: None explicitly mentioned
Negative control: None explicitly mentioned

Annotations

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