Protein concentrations in cell lysates were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein was separated by denaturing gel electrophoresis on NuPAGE 4-12% Bis-Tris Protein Gels using NuPAGE MES SDS running buffer. Protein was transferred onto PVDF membranes using a Trans-Blot Turbo Transfer System. Membranes were blocked in blocking buffer (tris-buffered saline with 0.1% Tween-20 (TBS-T 0.1%, 50 mM Tris, 150 mM NaCl at pH 7.6, 0.1% Tween-20 supplemented with 5% BSA in PBS) before overnight incubation in primary antibodies against HSP90 (27 ), beta-actin (28 ), phosphoTie2 (21 ), or insulin receptor beta subunit (29 ), diluted in blocking buffer at 4°C. Washes in 0.02% TBS-T were performed before incubation with a secondary antibody against mouse (30 ), or rabbit (31 ), diluted in blocking buffer or 0.02% TBS-T for 2 hours at room temperature. Membranes were imaged with a Syngene G:BOX after incubation with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore).
G box
Manufactured by Merck Group
The G:BOX is a versatile laboratory equipment designed for image acquisition and analysis. It features a high-resolution camera and advanced imaging software to capture and process a wide range of samples, including gels, blots, and chemiluminescent samples. The G:BOX provides researchers with a reliable and efficient tool for their laboratory workflow.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti p atm, by Cell Signaling Technology (1 mentions)
Anti p p53, by Cell Signaling Technology (1 mentions)
Anti gamma h2ax, by Cell Signaling Technology (1 mentions)
Anti actin, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti caspase 7, by Cell Signaling Technology (1 mentions)
Anti caspase 9, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
2 protocols using g box
1
Western Blot Analysis of Cellular Proteins
2
Quantifying DNA Damage Response Proteins
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AGO1552s were trypsinised off Mylar, lysed and protein concentration determined using the Bradford assay. Lysates were then run on 12% acrylamide gels (8% for p-ATM), transferred to a nitrocellulose membrane, blocked in 5% milk followed by incubation overnight at 4 0 C with antigamma-H2AX (see above), anti-p-ATM (see above), anti-Actin, anti-BCL-2, anti-Bax, anti-Caspase 3, anti-Caspase 7, anti-Caspase 9 and anti-p-p53 (Cell Signalling) primary antibodies.
Membranes were then washed in TBS-0.1% tween20 and incubated at room temperature for 1 h with HRP-conjugated secondary antibodies (Cell Signalling). Luminata (Merck Millipore) was used for chemiluminescence and membranes were visualised on a Syngene G:BOX with exposure time set automatically to prevent saturation. AGO1552 cells were used for western blotting to as the number of alpha particles delivered to each adhering fibroblast cell could be kept far more consistent than with lymphocytes in suspension.
Membranes were then washed in TBS-0.1% tween20 and incubated at room temperature for 1 h with HRP-conjugated secondary antibodies (Cell Signalling). Luminata (Merck Millipore) was used for chemiluminescence and membranes were visualised on a Syngene G:BOX with exposure time set automatically to prevent saturation. AGO1552 cells were used for western blotting to as the number of alpha particles delivered to each adhering fibroblast cell could be kept far more consistent than with lymphocytes in suspension.
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