Twenty-five micrograms of total protein extracts prepared according to standard methods were fractioned by SDS-PAGE (4–12% Bis–Tris/SDS polyacrylamide gel, NuPAGE, Thermo Fisher Scientific) and transferred onto Hybond nitrocellulose filters (GE Healthcare Life Sciences, Buckinghamshire, UK). Filters were blocked for 1 h at room temperature in 1× PBS-Tween20, 5% skim milk, and then incubated overnight at 4 °C with primary antibodies: Rabbit polyclonal anti-γ-H2AX (ab11174, Abcam, Cambridge, UK); anti-LAMP-1 (ab24170, Abcam); anti-LC3B (ab51520, Abcam); and anti-PARP-1 (#9542, Cell Signaling Technology, Danvers, MA, USA). Mouse monoclonal anti-GAPDH (G8796, Sigma-Aldrich S.r.l.) and anti-Vinculin (VCL, V9131, Sigma-Aldrich S.r.l.) antibodies were used to ensure equal protein loading. The filters were then probed with secondary, peroxidase-linked whole antibodies (GE Healthcare) for 1 h at room temperature, and blotted proteins were detected using the Novex® ECL HRP Chemiluminescent detection system (Thermo Fisher). Filters were then subjected to autoradiography, the films were scanned, and images were analyzed using ImageJ 1.46r.
Secondary peroxidase linked whole antibodies
Manufactured by GE Healthcare
Sourced in Italy
Secondary peroxidase-linked whole antibodies are laboratory reagents used in various immunoassay techniques, such as Western blotting and enzyme-linked immunosorbent assays (ELISAs). They are designed to bind to primary antibodies and facilitate the detection of target proteins or antigens. The peroxidase enzyme linked to these antibodies can be used to catalyze a chromogenic or chemiluminescent reaction, allowing for the visualization and quantification of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (4 mentions)
Ab11174, by Abcam (2 mentions)
Hybond nitrocellulose filters, by GE Healthcare (2 mentions)
Novex ecl hrp chemiluminescent detection system, by Thermo Fisher Scientific (2 mentions)
Anti parp1, by Cell Signaling Technology (2 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Ab51520, by Abcam (1 mentions)
Ab24170, by Abcam (1 mentions)
β actin monoclonal antibody, by Abcam (1 mentions)
Iblot device, by Thermo Fisher Scientific (1 mentions)
Visionworks ls software, by Analytik Jena (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ab7960, by Abcam (1 mentions)
Ab33899, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Sc 9271, by Santa Cruz Biotechnology (1 mentions)
Imagescanner 3, by GE Healthcare (1 mentions)
Anti myc, by Abcam (1 mentions)
5 protocols using secondary peroxidase linked whole antibodies
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Protein Quantification
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Protein levels were assessed by western blot analyses of whole cell lysates prepared according to standard procedures. Proteins (40 μg) were fractioned by SDS-PAGE and transferred onto Hybond nitrocellulose filters (GE Healthcare Italia, Milano, Italy). Filters were blocked in PBS-Tween 20/5 % skim milk and incubated overnight with primary antibodies (see above). The filters were then probed with secondary peroxidase-linked whole antibodies (GE Healthcare Italia) and bound antibodies visualized by SuperSignal® West PICO chemiluminescent detection system (Fisher Scientific, Pittsburgh, PA, USA). Filters were then subjected to autoradiography. An anti-β-actin (ACTB) antibody was used on each blot to confirm equal protein loading.
3
Characterization of RMS Cell Lines
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Protein expression of all RMS cell lines was also characterized by WB assay, and for those cell lines that resulted IHC ALK positive, its phosphorylated form was also investigated. Samples containing 200 μg of protein per lane were separated on precast 4–12% NuPAGE bis-tris gels (Invitrogen), and were transferred onto Hybond ECL nitrocellulose membranes (Invitrogen) using the NuPAGE transfer buffer and iBlot device (Invitrogen). Nitrocellulose membranes were blocked in PBS-Tween 20 with 5% skim milk, first incubated with the primary antibodies (Cell Signaling) and then with the secondary peroxidase linked whole antibodies (GE Healthcare Europe). Bound antibodies were detected using the Super Signal chemiluminescent substrate (GE Healthcare Europe). β-Actin monoclonal antibody (Abcam) was used to confirm equal protein loading on the gel. Filters were autoradiographed, and autoradiographs were scanned and quantified by densitometric analysis using Vision Works LS software (UVP, Upland, CA, USA). The H2228 lung adenocarcinoma cell line, positive for ALK rearrangement (EML4-ALK), was utilized as positive control for ALK expression (total protein and phosphorylated form at 80kDa).
4
Western Blot Analysis of Protein Targets
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Total protein extracts were prepared according to standard methods. Forty micrograms of protein extract was fractioned by SDS-PAGE and transferred onto Hybond nitrocellulose filters (RPN 303D, GE Healthcare, Milan, Italy). Filters were blocked in PBS-Tween 20 and 5% skim milk and incubated overnight with the following primary antibodies: mouse monoclonal anti-BCL-2 (sc-509, Santa Cruz Biotechnology, Dallas, TX, USA), anti-KIT (#3308; Cell Signaling Technology, Danvers, MT, USA), and anti-MYC (ab32; Abcam, Cambridge, United Kingdom); rabbit polyclonal anti-AKT(S473) (sc-9271, Santa Cruz Biotechnology), anti-BRAF (ab33899, Abcam,), anti-ERK1/2 (T202/Y204) (#9101, Cell Signaling Technology), anti-γ-H2AX (ab11174, Abcam), anti-p21waf1 (ab7960, Abcam), and anti PARP-1 (#9542, Cell Signaling Technology). Mouse monoclonal anti-Vinculin (VCL, V9131, Sigma-Aldrich, Milan, Italy) or anti-β-Actin (Ab8226, Abcam) antibodies were used to ensure equal protein loading. The filters were then probed with secondary peroxidase-linked whole antibodies (GE Healthcare, Milan, Italy) and subjected to autoradiography using the Novex® Enhanced Chemoluminescent Horseradish Peroxidase detection system (Thermo Fisher Scientific, Monza, Italy). Films were scanned (ImageScanner III, GE Healthcare, Milan, Italy) and images were processed by Photoshop7.0.1 or analyzed using ImageJ 1.46r.
5
Western Blot Analysis of Protein Markers
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Forty micrograms of total protein extracts prepared according to standard methods was fractioned by SDS-PAGE, transferred onto nitrocellulose filters and incubated o.n. with primary antibodies: mouse monoclonal antibodies raised against β–actin, PAX8, TNKS, WRAP53; rabbit polyclonal antibodies raised against H2AX, γ- H2AX (S139), p21Waf1, POT1, PRKCA (Y124), TEP1 and a goat polyclonal antibody raised against TNKS2 (Abcam, Cambridge, UK); rabbit polyclonal antibodies raised against TERF2, TSPYL5 and the mouse monoclonal raised against p53 (Santa Cruz Biotechnology); rabbit polyclonal antibodies raised against PARP1 and CPP32 (Cell Signaling Technology, Danvers, MA, USA). The filters were then probed with secondary peroxidase-linked whole antibodies (GE Healthcare, Milano, Italy) and detected by Novex® ECL HRP Chemiluminescent detection system (Thermo Fisher). Filters were then subjected to autoradiography. β-Actin (ACTB) was used on each blot to ensure equal protein loading.
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