Cells, tissues, and mouse xenograft tumors were harvested following various treatments as described above, lysed in NP-40 lysis buffer (Boston BioProducts), and quantified by a DC Protein Assay (Bio-Rad). For Western blot analyses, protein samples (10 mg) were separated on a NuPAGE 4%–12% Bis-Tris Protein Gel (Invitrogen) and transferred onto Immobilon-P PVDF membranes (EMD Millipore). The membranes were incubated for 1 hour in blocking buffer [5% nonfat dry milk in Tris-buffered saline and 0.1% Tween (TBS-T)], followed by incubation overnight at 4 C with the primary antibody. After three washes with TBS-T, the blots were incubated with horseradish peroxidase– conjugated secondary antibody, and signals were visualized by Luminata Forte Western blot HRP substrate (EMD Millipore) using an Amersham Imager 600RGB (GE Healthcare Life Sciences). Antibodies used are listed in Supplementary Table S3 .
Luminata forte western blot hrp substrate
Manufactured by Merck Group
Sourced in United States
Luminata Forte Western blot HRP substrate is a chemiluminescent detection reagent used in Western blot analysis. It is designed to produce a strong and consistent signal for the detection of horseradish peroxidase (HRP)-labeled target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Np 40 lysis buffer, by Boston BioProducts (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Amersham imager 600rgb, by GE Healthcare (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Facsaria, by BD (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Complete ultra protease inhibitor, by Roche (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Phenylmethylsulphonyl fluride, by Merck Group (1 mentions)
2 protocols using luminata forte western blot hrp substrate
1
Western Blot Analysis of Protein Samples
2
Protein Analysis from Spleen and Bone Marrow
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Western blots were performed according to standard procedures using protein lysates prepared from whole spleen samples or sorted cell populations from spleen or bone marrow (sorted using FACSAria, BD Biosciences) using RIPA buffer (300 mM NaCl, 2% IGEPAL CA-630, 1% deoxycholic acid, 0.2% SDS, 100 mM Tris-HCl pH 8.0) containing complete ULTRA protease inhibitors (Roche, Basel, Switzerland) and phenylmethylsulphonyl fluride (Sigma-Aldrich). Samples were run on NuPAGE Bis-Tris 10% gels (Life Technologies, Carlsbad, CA, USA) with NuPAGE MOPS SDS running buffer (Life Technologies) and transferred to nitrocellulose membranes with an iBlot (Life Technologies). Blots were probed with antibodies to Mcl-1 (clone 19C4-15, WEHI monoclonal facility), Flag (clone 11F3, WEHI monoclonal facility) and β-actin (clone AC-74, Sigma, catalogue #A2228), used as a loading control, followed by secondary staining with horseradish peroxidase (HRP)-conjugated rat or mouse Ig-specific secondary antibodies (Southern Biotech) and visualized using Luminata Forte western blot HRP substrate (Merck Millipore, Billerica, MA, USA) on a ChemiDocTM Touch Imaging System (Bio-Rad, Hercules, CA, USA).
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