Keratinocytes were plated directly in six-well plates in KGM with supplements. Following the indicated treatments, the cells were lysed and subjected to Western blot as previously described [79 (link)] with minor modifications. The transfer of protein to a nitrocellulose membrane (AmershamTM Protran 0.45 lm NC; GE Healthcare) was achieved at 25 V overnight at 4 °C. The membrane was then blocked with 5% (w/v) BSA in TBS-T at pH 7.2 for 1 h before being incubated with mouse monoclonal IgG2a XPC (Santa Cruz Biotechnologies, CA, USA) at 1 μg/mL in 5% (w/v) BSA in TBS-T, overnight at 4 °C. The following day, the membrane was washed with TBS-T three times and incubated with secondary goat anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA) in 5% (w/v) BSA in TBS-T for 1 h at room temperature. After incubation, the membrane was washed three times with TBS-T before adding chemiluminescence substrate (Millipore SAS, Molsheim, France) for band detection. The bands were imaged with the ChemiDocTM imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at the Bosch Research Institute (University of Sydney), and densitometry was carried out using ImageJ software (version 1.50).
Secondary goat anti mouse igg hrp linked antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The secondary goat anti-mouse IgG HRP-linked antibody is a reagent used in immunoassays and other immunochemical techniques. It is a polyclonal antibody that binds to mouse immunoglobulin G (IgG) and is conjugated with horseradish peroxidase (HRP) enzyme, which can be detected through colorimetric or chemiluminescent methods.
Automatically generated - may contain errors
2 protocols using secondary goat anti mouse igg hrp linked antibody
1
Western Blot Analysis of XPC
2
Quantitative Western Blot Analysis
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Cultures for total protein extraction were grown in MM supplemented with urea at 25°C for 16 h. Total protein extraction was performed as previously described (Galanopoulou et al., 2014) . Equal sample loading was estimated by Bradford assays and Coomasie staining. Total proteins (30 μg) were separated by SDS-PAGE (10% w/v polyacrylamide gel) and electroblotted (Mini PROTEAN™ Tetra Cell, BIORAD) onto PVDF membranes (Macherey-Nagel, Lab Supplies Scientific SA, Hellas) for immunodetection. The membrane was treated with 2% (w/v) non-fat dried milk, and immunodetection was performed with a primary mouse anti-GFP monoclonal antibody (Roche Diagnostics, Hellas), a mouse anti-actin monoclonal (C4) antibody (MP Biomedicals Europe) and a secondary goat anti-mouse IgG HRP-linked antibody (Cell Signaling Technology Inc, Bioline Scientific SA, Hellas). Blots were developed by the chemiluminescent method using the LumiSensor Chemiluminescent HRP Substrate kit (Genscript USA, Lab Supplies Scientific SA, Hellas) and SuperRX Fuji medical X-Ray films (FujiFILM Europe, Lab Supplies Scientific SA, Hellas). Densitometry analysis of Fur-GFP, free-GFP and actin-specific bands was performed using the ImageJ software (Schneider et al., 2012) . The relative density of the Fur-GFP and free-GFP bands were quantified and normalised by dividing with the respective actin-band density.
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