24 hours post irradiation, cells were washed once with PBS and scraped and harvested in sample buffer (2.5% w/v SDS, 25% v/v Glycerol, 125mM Tris pH-6.8, 0.01% w/v bromophenol blue, 4% β-mercaptoethanol, in water). Samples were sonicated for ten seconds (30% amplitude, Branson Digital Sonifier) and heated at 95°C for 5 minutes. Proteins were resolved on 10% SDS-polyacrilamide gels and transferred to 0.45μm nitrocellulose membranes (Bio-Rad). Western blots were probed with anti-mouse p53 (Cell signaling) and GAPDH (Millipore). Antibodies were detected using peroxidase-conjugated AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch Labs) and enhanced chemiluminescence western blotting detection reagents (Thermo Fisher scientific). Gels were Imaged by CemiDoc XRS+ (Bio-Rad) and analyzed by ImageLab 5.1 software (BioRad).
Cemidoc xrs
Manufactured by Bio-Rad
The ChemiDoc XRS+ is a compact, high-performance imaging system designed for diverse imaging applications in the life sciences laboratory. It features a cooled, high-resolution CCD camera, a high-efficiency transillumination system, and a motorized lens to capture detailed images of a variety of sample types, including gels, membranes, and microplates.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence western blotting detection reagent, by Thermo Fisher Scientific (1 mentions)
Digital sonifier, by Emerson (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Mouse anti p53, by Cell Signaling Technology (1 mentions)
0.45 μm nitrocellulose membrane, by Bio-Rad (1 mentions)
Affinipure goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Gapdh, by Merck Group (1 mentions)
Ab62352, by Abcam (1 mentions)
Westernbright sirius, by Advansta (1 mentions)
Sc 25280, by Santa Cruz Biotechnology (1 mentions)
Sonopuls gm mini20, by Bandelin (1 mentions)
2 protocols using cemidoc xrs
1
Western Blot Analysis of p53 in Irradiated Cells
2
Quantification of Nrf2 Protein in Islet Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Approximately 400 islets per condition were lysed with RIPA buffer (mmol/L): 65 TRIS, 150 NaCl, 0.9 EDTA, and 1% Nonidet-P40 (10%), containing 1% protease inhibitor cocktail (freshly added) and 0.1% dithiothreitol. Samples were homogenized by an ultrasonic homogenizer (SonoPuls GM mini20, BANDELIN) for 30 s, and protein content was determined by a Bradford assay. Samples were diluted to a standardized protein concentration. Proteins were separated on a 10% polyacrylamide gel and blotted on a nitrocellulose membrane (Amersham™ Protan®, VWR, Germany), followed by incubation with anti-Nrf2 primary antibody (Abcam, ab62352, 1 : 1000) at 4°C up to 48 h. PCNA protein was used as the loading control (Santa Cruz, sc-25280 1 : 500). The secondary antibody (Cell Signaling Technology, anti-rabbit, No. 7074 1 : 1000) was added at room temperature for 1 h. All antibody dilutions were done with buffer containing 5% nonfat dry milk. Chemiluminescence (WesternBright™ Sirius™, Advansta Inc.) was detected with CemiDoc™ XRS (Bio-Rad).
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