Proteins were resolved on an SDS-PAGE gel. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (EMD Millipore) and then blocked in a blocking solution (1X Tris-buffered saline (TBS: 10 mM Tris base and 150 mM NaCl) with a pH of 7.4 and 10% blocking reagent (Roche BM Chemiluminescence Western Blotting Substrate)) for 1 hour at room temperature. The membrane was then incubated in primary antibody in TBS with 0.01% Tween (TBST) and 5% blocking reagent overnight at 4°C. The α-Tubulin, pGSK3β, GSK3β, β-actin, AKT, Lamin A/C, pS6K, S6K, p4E-BP1, 4E-BP1, and eIF4E antibodies were all purchased from Cell Signaling Technology. The BRD7 antibody was produced by Covance Inc. Next, the membrane was washed in TBST 3 times for 20 minutes followed by incubation in secondary antibody in TBST with 5% blocking reagent for 1 hour at room temperature. Goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology and anti-rabbit IgG HRP-linked antibody was purchased from Cell Signaling Technology. The membranes was washed 3 times in TBST for 20 minutes each wash. Membranes were developed using either Thermo Scientific West Femto Maximum Sensitivity Substrate or Roche BM Chemiluminescence Western Blotting Substrate depending on the strength of the immunoblotted protein. Denville Scientific HyBlot CL Film was used to develop membranes.
Bm chemiluminescence western blotting substrate
Manufactured by Roche
Sourced in Germany
The BM chemiluminescence western blotting substrate is a reagent used in western blot analysis to detect and quantify proteins. It generates a chemiluminescent signal when it interacts with the protein-bound enzyme, allowing for the visualization and analysis of specific proteins in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Hyblot cl film, by Thomas Scientific (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
Eif4e, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
P s6k, by Cell Signaling Technology (1 mentions)
Lamin a c, by Cell Signaling Technology (1 mentions)
4e bp1, by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
P 4ebp1, by Cell Signaling Technology (1 mentions)
X ray sensitive film, by Roche (1 mentions)
Tgx stain free precast gel, by Bio-Rad (1 mentions)
Stain free precast gel, by Bio-Rad (1 mentions)
Clone 3f10, by Roche (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
4 protocols using bm chemiluminescence western blotting substrate
1
Western Blot Protein Detection
2
Western Blot Protein Analysis
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Total protein lysates were loaded equally in the wells of SDS-PAGE, and after electrophoresis, transferred to PVDF membrane (Millipore). Thereafter, the membranes were washed with TBS solution and blocked for 30 min in 5% free fat milk dissolved in 1X TBST (Tris-base, NaCl and 0.001% Tween-20 with pH 7.6). After blocking, the membranes were incubated overnight at 4°C with the corresponding primary antibody (Table S1 ) diluted in the same blocking solution. POD-conjugated secondary antibody and BM chemiluminescence western blotting substrate (Roche) were used to visualize bands on an X-ray sensitive film (Roche). Western blots were repeated at least three times and a representative blot from consistent triplicates was chosen for the figures. The graphs were prepared to represent the intensity of the bands on the x-Ray films which were quantified using the Image J software and the average of three independent replicates was calculated.
3
Western Blot Analysis of p53 in LV Myocardial Samples
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The Western blot analysis of p53 in LV myocardial samples was performed as described previously (Kollárová-Brázdová et al., 2021 (link)). Briefly, proteins in the LV myocardial samples were separated by SDS-PAGE using TGX Stain-Free precast gels (Bio-Rad, Hercules, CA). Immunodetection was performed with a mouse anti-p53 purified primary antibody (BP53-12; Exbio Praha a.s., Prague Czech Republic; dilution 1:1000) and an anti-mouse secondary antibody (P0447, Polyclonal Goat Anti-Mouse Immunoglobulin/HRP; DAKO Denmark A/S, Glostrup, Denmark; dilution 1:1000). A BM Chemiluminescence Western blotting Substrate (Roche) and Fusion Solo S imager coupled with a CCD camera (Vilber Lourmat GmbH, Eberhardzell, Germany) were used for signal detection. The results were normalized based on the total protein levels observed on the Stain-Free precast gels (Bio-Rad).
4
Western Blot Analysis of Protein Targets
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Cells were harvested and washed in PBS supplemented with protease inhibitor cocktail Set III (1:100) (CalBioChem). Cell lysate preparation, SDS–PAGE, and transfer were conducted as previously described (Concepción-Acevedo et al., 2012 (link)). Membranes were incubated in 1% Roche blocking reagent (1 h) followed by incubation with antibodies diluted in 0.5% blocking reagent (1 h). Peroxidase–anti-peroxidase soluble complex reagent (1:2000, Sigma) was used for PTP-tag detection. Rat monoclonal anti-HA (1:1000, 3F10 clone, Roche) and goat anti-rat (1:1000, Sigma) were used for HA detection. For subsequent detections, membranes were stripped with 0.1 M glycine (pH 2.5, 15 min, 37°C), washed in TBS (0.1% Tween-20), and blocked and reprobed with one of the following primary/secondary antibody combinations: C. fasciculata anti-Hsp70 (1:5000) (Johnson and Englund, 1998 )/chicken anti-rabbit (1:2000, Roche), T. brucei anti-Pol β (1:1000) (Saxowsky et al., 2003 (link))/goat anti-rat (1:5000) and anti-TAO (T. brucei alternative oxidase; 1:100) (Chaudhuri et al., 1998 )/goat anti-mouse (1:1000), and anti-tubulin (1:20,000, Sigma)/goat anti-mouse (1:1000). All secondary antibodies were horseradish peroxidase conjugated. Signal was detected with BM Chemiluminescence Western Blotting Substrate (POD) from Roche.
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