Lysates for immunoblotting were prepared from frozen cell pellets or snap-frozen xenograft tumors using RIPA buffer (Santa Cruz Biotechnology) with 1% Protease Inhibitor Cocktail (Sigma-Aldrich). Protein concentrations were determined using Bradford methodology (Bio-Rad). Proteins (50 μg per load) were resolved onto a 4–20% SDS-PAGE gradient gel (Bio-Rad), transferred onto a nitrocellulose membrane, blocked with 5% milk for 2 hrs, then incubated in primary antibody at 4 °C overnight (ZEB1 H-102, 1:200, sc-25388; ZEB2 H-260, 1:200, sc-48789; E-cadherin H-108, 1:500, sc-7870; N-cadherin, 1:500, ab18203; Vimentin V9, 1:200, sc-6260; β-Actin AC-15 1:10,000, sc-69879). All primary antibodies were obtained from Santa Cruz Biotechnology except for the anti N-cadherin antibody, which was obtained from AbCam. Membranes were then washed in TBST and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:5000, GE Healthcare) for 1 h at room temperature. Blots were developed and visualized using the Amersham™ ECL™ Western Blotting Detection Reagent kit (GE Healthcare Life Sciences). Antibody for β-actin was used as a loading control.
Amersham ecl western blotting detection reagent kit
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The Amersham™ ECL™ Western Blotting Detection Reagent kit is a chemiluminescent detection system used in western blotting to visualize and analyze protein expression levels in biological samples. The kit contains reagents required for the detection process.
Automatically generated - may contain errors
Lab products found in correlation
Criterion xt bis tris precast gel 4 12, by Bio-Rad (2 mentions)
Trans blot turbo blotting system, by Bio-Rad (2 mentions)
Anti n cadherin antibody, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse igg, by GE Healthcare (1 mentions)
Gradient sds page gel, by Bio-Rad (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
4 protocols using amersham ecl western blotting detection reagent kit
1
Immunoblotting Assay for Epithelial-Mesenchymal Transition
2
Western Blot Analysis of mNG Protein
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Cells were washed once in PBS. Laemmli loading buffer was added to the cells, and samples were boiled for 5 min. 20 µg protein was loaded into each lane of a Criterion XT Bis-Tris precast gel 4–12% (Bio-Rad Laboratories) for SDS-PAGE separation. XT-MOPS (1×) diluted in deionized water was used as a running buffer. Proteins were transferred onto nitrocellulose membranes using the Trans-Blot Turbo blotting system (25 V over 7 min; Bio-Rad Laboratories). The membrane was blocked with 5% skimmed milk for 1 h and then incubated with the monoclonal anti-mNG (32F6) primary antibody (ChromoTek) diluted 1:1,000 in 0.05% PBS–Tween-20 (PBST). As a loading control, the anti-PFR L13D6 monoclonal antibody (Kohl et al., 1999 (link)) diluted 1:25 was used. After primary antibody incubation, three washes of 5 min each were performed in 0.05% PBST followed by secondary antibody incubation. Anti-mouse secondary antibody was coupled to HRP and diluted to 1:20,000 in 0.05% PBST containing 0.1% milk, and then the membrane was incubated with this for 1 h. The Amersham ECL Western blotting detection reagent kit (GE Healthcare) was used for final detection of proteins on the membrane.
3
Osteoclastogenesis Pathway Regulation
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BMMs were seeded in 6-well plates at a density of 4 × 105 cells per well with 30 ng/mL M-CSF, 50 ng/mL RANKL and BCPA at the indicated concentrations. After 4 days, the cells were scraped for whole protein extraction. The proteins were electrophoretically separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with Tris buffered saline-Tween (TBST) containing 5% skim milk for 1 h and incubated overnight at 4 °C with Pin1 (sc-46660; Santa Cruz Biotechnology, Santa Cruz, CA, USA), DC-STAMP (MABF39-I, Merck Millipore, Darmstadt, Germany) and β-actin (A5441; Sigma-Aldrich) primary antibodies. The membranes were washed four times for 5 min each time with TBST buffer and incubated with a secondary horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology). The reaction was detected using an Amersham ECL Western Blotting Detection Reagent kit (RPN2106; GE Healthcare, Chicago, IL, USA) and pictures were taken with an EZ-Capture MG device (ATTO, Tokyo, Japan).
4
Western Blot Protocol for IFT172 Detection
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Cells were washed once in Phosphate Buffer Saline (PBS). Laemmli loading buffer was added to the cells and samples were boiled for 5 minutes. 20μg of protein were loaded into each lane of a Criterion™ XT Bis-Tris Precast Gel 4-12% (Bio-Rad, UK) for SDS-Page separation. XT-Mops (1X) diluted in deionised water was used as a running buffer. Proteins were transferred onto nitrocellulose membranes using the BioRad â Trans-Blot Turbo TM blotting system (25V over 7 minutes). The membrane was blocked with 5% skimmed milk overnight and then incubated with the monoclonal anti-IFT172 antibody (Absalon et al., 2008) diluted 1/500 in 0.1% milk and 0.1% Tween in PBS (PBST). As a loading control the anti-PFR L13D6 (Kohl et al., 1999) and 2E10 (Ismach et al., 1989 ) monoclonal antibodies were used. After primary antibody incubation, three washes of 5 minutes each were performed in 0.05% PBST followed by secondary antibody incubation. Anti-mouse secondary antibody coupled to horseradish peroxidase, diluted to 1/20,000 in 0.05% PBST containing 0.1% milk, and the membrane was incubated with this for 1 hour. The Amersham ECL Western Blotting Detection Reagent Kit (GE Healthcare Life Sciences, UK) was used for final detection of proteins on the membrane.
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