After treatment, cells were quickly rinsed with cold PBS (1 ml). Cells were collected by a rubber scraper in lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 5 mM β-glycerophosphate, 1 mM MgCl2, 1% Triton X-100, 1 mM sodium orthovanadate, 10 μg/ml protease inhibitors, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. The cells were destroyed by sonication on ice for 12 s. Cell debris were removed by centrifugation at 4 °C at 5000 rpm for 10 min. Protein concentrations were measured using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of samples were loaded to SDS-PAGE gel and transferred to membrane. Blots were blocked with 1% of BSA in TBST and immunoblotted with primary antibodies for 2 h (room temperature) or overnight (4 °C). The membranes were then washed for 10 min x 3 times with TBST. Secondary antibodies were added for a further 1 h. Immunosignals were stimulated by an Enhanced Chemiluminescence Detection Kit (Thermo Fisher Scientific, Waltham, MA) and recorded with an Azure c600 imaging system.
Azure c600 imaging system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Azure c600 imaging system is a compact and versatile instrument designed for various imaging applications. It features a high-resolution camera and advanced illumination options to capture clear and detailed images. The system is capable of performing different imaging techniques, but a detailed description of its core function and intended use is not available without potential bias or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay kit, by Bio-Rad (1 mentions)
Enhanced chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
Nupage mops running buffer, by Thermo Fisher Scientific (1 mentions)
Rabbit anti ha tag, by Cell Signaling Technology (1 mentions)
Rabbit trueblot anti rabbit igg hrp, by Rockland Immunochemicals (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail set 3, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Immobilon p polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Adi sab 300, by Enzo Life Sciences (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Bio safe coomassie stain, by Bio-Rad (1 mentions)
4 protocols using azure c600 imaging system
1
Cell Lysis and Protein Analysis
2
Immunoblot Analysis of Tagged Proteins
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Cells were lysed with ice cold iCLIP Lysis Buffer3 (link) containing 1X Protease Inhibitor Cocktail Set III (Milipore Sigma, Cat # 539134-1SET). Lysates were sonicated for 5 min at 30 second on/off intervals and protein was quantified using Pierce BCA Protein Assay Kit (Thermo Fisher, Cat # 23225). Total protein lysates were run on 4%–12% NuPAGE Bis-Tris gels in NuPAGE MOPS running buffer (Thermo Fisher, Cat # NP0050) and transferred to PVDF membranes. Membranes were blocked in 5% nonfat milk in TBST and incubated with the following primary antibodies: 1 h at room temperature with rabbit anti-HA-tag (Cell Signaling, Cat # 3724, Clone C29F4, Lot 10), washed 3X for 5 min with TBST, incubated for 2 h at RT in 5% nonfat dry milk powder in TBST with Rabbit TrueBlot: Anti-Rabbit IgG HRP (Rockland Immunochemicals, Cat # 18-8816-33, Clone eB182, Lot 46967), washed 3X for 5 min with TBST. Membranes were developed using the Azure C600 imaging system and Pierce ECL Western Blotting Substrate (Thermo Fisher, Cat # 32209).
3
Western Blot Analysis of EGFP Expression
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Twenty-four hours post-transfection with DNA encoding the indicated plasmid, N2As were lysed in 2× protein sample buffer (150 µL). Cell lysates were subjected to SDS-PAGE, transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA); after a 60 min, incubation in blocking solution (2% in 0.1% TBST) membranes were incubated with rat α-EGFP-monoclonal IgG (3H9, Chromotek, Planegg, DE, 1:5000 final dilution) or mouse α-β-actin monoclonal IgG (A2228, Millipore Sigma, Mass, USA, 1:20,000 final dilution). Bound primary antibodies were recognized as α-rat or α-mouse IgG secondary antibody conjugated with horseradish peroxidase (Life Technologies, MA, USA, 1:1000), and secondary antibodies were visualized using Super Signal™ West Pico PLUS Chemiluminescent Substrate (34578, Thermo, IL, USA) on an Azure C600 Imaging System (Dublin, CA, USA), using a 60 s exposure.
4
Protein Separation and Western Blotting
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Proteins were separated by SDS-PAGE using 4–20% or 7.5% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, Hercules, CA) followed by detection with Bio-Safe Coomassie Stain (Bio-Rad). For Western immunoblotting experiments, proteins in SDS-PAGE gels were transferred onto a PVDF membrane at 4°C using Mini Trans-Blot Cell (Bio-Rad), blocked with 5% nonfat milk for 1 hr at room T or overnight at 4°C and incubated with the following primary antibodies at indicated dilutions for 1 hr at room T: 1:10,000 HSF1 polyclonal antibody (Enzo Life Sciences ADI-SPA-901-F; Lausen, Switzerland), 1:4000 p53 rabbit antibody (SUMOlink SUMO-1 Kit), 1:5000 SUMO-1 rabbit antibody (SUMOlink SUMO-1 Kit). Secondary antibody incubation was performed for 1 hr at room T using 1:10,000 goat anti-rabbit IgG polyclonal antibody (Enzo Life Sciences, ADI-SAB-300). Blots were developed using SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, Waltham, MA) and imaged using the Azure c600 Imaging System.
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