For immunoblotting, cells were lysed using RIPA buffer supplemented with 1 mM Na3VO4 (Santa-Cruz Biotechnology, Dallas, USA) and a protease inhibitor cocktail (P8340, Sigma-Aldrich, St.Louise, Missouri, USA). DC Protein Assay (Bio-Rad, Hercules, USA) was employed to assess protein concentration, using bovine serum albumin as the standard. Protein samples were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Amersham™ Protran™ Premium, GE Healthcare, USA). Blocking was performed by incubating membranes for 1 h with 5% BSA in TBS-T (0.1% Tween-20) and incubated O/N at 4 °C with primary antibodies. The full list of the antibodies used is provided in Supplementary table 1 . Eventually, membranes were incubated for 1 h with horseradish peroxidase-coniugated secondary antibodies (mouse or rabbit) and protein bands were detected by chemiluminescence (Amersham™ ECL™ Detection Reagents).
Amersham protran premium
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
Amersham Protran Premium is a high-performance nitrocellulose membrane designed for protein transfer and detection applications. It offers consistent pore size, thickness, and binding capacity to ensure reliable and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Na3vo4, by Santa Cruz Biotechnology (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Phosphatase inhibitor cocktail, by Nacalai Tesque (2 mentions)
Amersham ecl select, by GE Healthcare (2 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
P8340, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ponceau s solution, by Merck Group (1 mentions)
X omat ls film, by Kodak (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Anti phospho akt thr308 rabbit mab, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti phospho akt ser473 antibody, by Cell Signaling Technology (1 mentions)
7 protocols using amersham protran premium
1
Immunoblotting Protocol for Protein Analysis
2
Western Blot Protein Detection
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Starved and treated cells were lysed using RIPA buffer supplemented with a protease inhibitor cocktail (P8340, Sigma-Aldrich) and 1 mM Na3VO4 (Santa-Cruz Biotechnology, Dallas, USA). Protein concentration was determined by DC Protein Assay (Bio-Rad, Hercules, USA), using bovine serum albumin as the standard. Protein samples were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Amersham™ Protran™ Premium, GE Healthcare, USA). Membranes were blocked for 1 h using 5% BSA (Sigma-Aldrich) in TBS-T (0.1% Tween-20) and incubated O/N (4 °C) with primary antibodies diluted in the blocking solution. Finally, membranes were incubated with horseradish peroxidase-coniugated secondary antibodies against mouse or rabbit for 1 h and protein presence was detected by chemiluminescent reaction (Amersham™ ECL™ Detection Reagents). Bands quantification was performed using Image Lab software. Details about the antibodies employed for proteins detection are reported in Supplementary Information .
3
Quantifying Protein Abundance in Plants
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Total protein extracts were prepared from 4‐week‐old plants by grinding leaves in Laemmli buffer and boiling samples for 5 min. The same amount of protein extracts were migrated on electrophoresis gel (SDS‐PAGE) before being transferred to Amersham™ Protran Premium nitrocellulose membranes (GE Healthcare, Chicago, IL). Membranes were then stained with Ponceau S solution (Sigma‐Aldrich) to assess equal loading and correct transfer. Incubation with antibodies directed against actin (1 : 5000 dilution) or eIF4E (1 : 2000 dilution, obtained from Estevan et al., 2014 ) was performed. Secondary antibody incubation was carried out using goat horseradish peroxidase‐labelled anti‐mouse serum for actin and anti‐rabbit serum for eIF4E from Sigma‐Aldrich at the same dilutions as the primary antibodies. Peroxidase activity was then checked using LumiGLO Reserve chemiluminescent substrate kit (KPL, www.kpl.com ) and X–OMAT LS films (Kodak, Rochester, NY).
4
Detailed Western Blot Methodology
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For Western blot (WB), proteins were extracted as previously described in (Stanisz et al, 2014 (link)). Shortly, 40–75 μg of protein were resolved on a 10% SDS–polyacrylamide gel and transferred onto a 0.45 μm nitrocellulose membrane (#10600003, Amersham Protran Premium, GE Healthcare). After transfer, membranes were blocked in 5% BSA or 5% skim milk powder solution, followed by an overnight incubation with primary antibodies. Membranes were incubated with secondary antibodies for 1 h in the dark at room temperature. Imaging and quantification of the blots was performed using an Odyssey infrared imaging system (LI‐COR, Lincoln, Nebraska, USA). Primary and secondary antibodies used for immunoblotting are listed in Tables EV4 and EV5 .
5
Immunoblot Analysis of Cell Lines
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For immunoblot analysis, MCF-7 and MDA-MB-175-VII cells were washed with PBS and lysed with lysis buffer containing a Phosphatase Inhibitor Cocktail (Nacalai Tesque Inc., Kyoto, Japan). Proteins were separated via SDS-PAGE and then electro-transferred onto nitrocellulose membranes (Amersham Protran Premium, GE Healthcare, Buckinghamshire, UK). The membranes were probed with various primary and secondary antibodies (Online Resource 1B) and visualized with enhanced chemiluminescence detection reagents (Amersham ECL Select, GE Healthcare, Buckinghamshire, UK). All western blotting experiments were performed in triplicate.
6
Immunoblotting of Heparin-Binding Proteins
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Enriched heparin binding proteins were centrifuged to pellet debris, separated by NuPAGE Novex 4–12% [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane gels (Thermo Fisher Scientific, Bremen, Germany), transferred to a nitrocellulose membrane (Amersham Protran Premium, GE Healthcare Life Sciences, Solingen, Germany), and analysed by immunoblotting. CCL16 was detected by a biotinylated polyclonal goat anti-human CCL16 antibody and streptavidin-coupled horseradish peroxidase (both R&D Systems, Wiesbaden, Germany). Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Solingen, Germany) was used as a substrate.
7
Immunoblot Analysis of Wnt5a Signaling
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For immunoblot analysis, MCF-7 normal, MCF-7/Wnt5a(+), and MCF-7/Wnt5a(-) cells were washed with PBS and lysed with lysis buffer containing a Phosphatase Inhibitor Cocktail (Nacalai Tesque Inc., Kyoto, Japan). Proteins were separated on an SDS-PAGE gel. The proteins were transferred onto a nitrocellulose membrane (Amersham Protran Premium, GE Healthcare, Buckinghamshire, UK) after electrophoresis. The membranes were probed with various primary and secondary antibodies and visualized with enhanced chemiluminescence detection reagents (Amersham ECL Select, GE Healthcare, Buckinghamshire, UK). All western blotting experiments were performed in triplicate. Anti-Wnt5a/b rabbit monoclonal antibody (mAb) (Cat #2530), anti-phospho-AKT (Ser473) antibody (Cat #9271), anti-phospho-AKT (Thr308) rabbit mAb (Cat #4056), and anti-phospho-SAPK/JNK antibody (Cat # 9251) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-ER (Cat #ab16660) and anti-HER2 antibodies (Cat #ab214275) were procured from Abcam (Cambridge, UK). Anti-β-actin antibody was purchased from Sigma-Aldrich (Cat #A5441, St Louis, MO, USA).
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