WT cells were grown exponentially in AB medium. At OD450 = 0.05, cells were treated with PNA-peptide (8 μM) and harvested after 3 h. Cells were washed in 10 mM Tris-HCl pH 8/10 mM MgCl2. Samples were heated at 95°C for 5 min and sonicated using a Branson 450 Sonifier. Protein content was determined using Bradford Reagent (Sigma–Aldrich) and normalized. Total protein (1.25 μg) was separated using SDS-PAGE (Precast Gel: 10%–20% Tris-HCL; Bio-Rad) in an XCell4 SureLock™ Midi-Cell (ThermoFisher Scientific) and transferred to a PVDF membrane (GE Healthcare, Whatman™), using a semi-dry blotting apparatus (JKA Biotech, Denmark). NrdA protein was detected with a rabbit polyclonal anti-NrdA antibody (MyBiosource) and goat anti-rabbit immunoglobulins/HRP (Agilent) as the secondary antibody. The PVDF membrane was incubated with ECL chemiluminescence substrate (Bio-Rad) and the signal detected using an ImageQuant LAS400 (GE Healthcare, Life Sciences).
Ecl chemiluminescence substrate
Manufactured by Bio-Rad
Sourced in United States
ECL chemiluminescence substrate is a laboratory reagent used to detect and quantify proteins in Western blot analysis. It is a luminol-based solution that reacts with horseradish peroxidase (HRP) conjugated to secondary antibodies, producing a luminescent signal proportional to the amount of target protein present. This substrate enables sensitive and quantitative detection of protein targets.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Goat anti rabbit immunoglobulins hrp, by Agilent Technologies (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Imagequant las 400, by GE Healthcare (1 mentions)
Whatman, by Cytiva (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti vinculin, by Abcam (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Merck Group (1 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Alpha tubulin, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Image lab software version 5, by Bio-Rad (1 mentions)
β tubulin, by Merck Group (1 mentions)
Renilla luciferase, by Thermo Fisher Scientific (1 mentions)
4 protocols using ecl chemiluminescence substrate
1
Quantifying NrdA protein expression
2
Western Blot Analysis of IkBα
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Total protein lysate (20ug) extracted from pre and post exercised muscle were separated on a 4–12% Bis Tris gel (Thermo Fisher Scientific, Waltham, MA, USA) and transferred for 2 hours at 4°C onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked for 1 hour using 5% BSA in TBS-0.1% Tween and incubated overnight at 4°C with IκBα (L35A5) mouse monoclonal antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA). Membranes were then washed with TBS-0.1% Tween and probed with anti-mouse IgG, HRP-linked antibody (1:5000; Cell Signaling Technology, Danvers, MA, USA) for 1 hour. ECL chemi-luminescence substrate (Bio-Rad, Hercules, CA, USA) was used to develop blot on ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA) using the optimal exposure settings. Densitometry analysis was carried out using Image J and ratios of the optical density were normalized to the corresponding loading control (anti-vinculin, 1:1000, Abcam Inc, Cambridge, MA, USA).
3
Protein Expression Analysis in Prostate Cancer
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Total proteins were extracted from C4‐2B‐30e, 22Rv‐1‐30e and PC‐3‐30e stable sublines at 24 and 48 h post induction with doxycycline using RIPA lysis buffer supplemented with phosphatase and protease inhibitors (Fisher Scientific). 50 μg of extracted proteins were separated on a 10% Bis‐Tris gel, transferred to activated PVDF membranes and were probed with primary antibodies specific to AR, PARP, cleaved caspase‐3 and CCND1 (Cell Signaling Technology) and PCNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Alpha‐tubulin (Cell Signaling Technology, Danvers, MA, USA) and GAPDH (Sigma Aldrich, St. Louis, MO, USA) antibodies were used as internal controls. ECL Chemiluminescence substrate (BioRad, Hercules, CA, USA) was used to develop the blots which were imaged using a ChemiDoc MP Imaging System (BioRad). Comparative expression was performed based on densitometry analysis using imagej [30 (link)] normalized to internal controls.
4
SDS-PAGE and Western Blot Analysis
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were conducted as previously described [26 (link), 27 (link)] with the following modifications. Briefly, whole cell lysates from plasmid-transfected HEK293T cells were harvested using Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA, USA) then separated using 12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Antibodies against Renilla luciferase (Thermo Fisher Scientific), β-tubulin (Sigma-Aldrich) and anti-PRRSV-N monoclonal antibody generated in-house (clone No. 6D10) were used for specific protein detection. Detection of primary antibody binding to targets was conducted by incubation of membranes with goat anti-rabbit IgG-conjugated or anti-mouse IgG-conjugated horseradish peroxidase (Sigma-Aldrich) and visualized after addition of ECL chemiluminescence substrate (Bio-Rad Laboratories). The chemiluminescence signal was recorded digitally using a ChemiDoc MP imaging system (Bio-Rad Laboratories). Digital signal acquisition and densitometry analyses were conducted using ImageLab Software, Version 5.1 (Bio-Rad Laboratories).
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