All strains were grown in GMM+YE overnight at 37 °C with agitation at 250 rpm. The resulting fungal mass was filtered and rinsed with sterile, deionized water, and crushed with liquid nitrogen. The macerated hyphal material was resuspended in 1:1 volumes of extraction buffer (25 mM Tris-HCl [pH 7.5], 10mM MgCl2, 150 mM NaCl, 1 mM EDTA, 0.01% NP-40, 2% glycerol, 1 mM Pefabloc [Sigma], 1 mM protein inhibitor cocktail [Sigma]), and the resulting crude lysates were centrifuged at 3,500 rpm for 9 min at 4°C. Cleared lysates were then transferred to new tubes and the total protein concentration was quantified using the Bradford assay. Fifty milligrams of cleared lysate were boiled before SDS-PAGE separation. Membranes were probed with the anti-Ras, clone RAS10 mouse monoclonal primary antibody (1:2000 dilution, EMD Millipore) and followed by the secondary antibody, a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG2a (1:2000 dilution, Abcam). Blots were imaged using a Bio-Rad ChemiDoc XRS HQ System and QuantityOne software (v4.6.5, Bio-Rad). The assay was performed in biologic triplicate for each strain.
Chemidoc xrs hq system
Manufactured by Bio-Rad
The ChemiDoc XRS HQ System is a compact, high-quality imaging system designed for capturing and analyzing chemiluminescent, fluorescent, and colorimetric gel and blot images. The system features a high-resolution camera and a range of illumination options to accommodate various imaging applications. It is a versatile tool for researchers in the life sciences and molecular biology fields.
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Lab products found in correlation
2 protocols using chemidoc xrs hq system
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Ras Protein Extraction and Western Blot
2
Western Blot Analysis of Fungal Ras Protein
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All strains were grown in GMM + YE overnight at 37°C with agitation at 250 rpm. The resulting fungal mass was filtered and rinsed with sterile, deionised water and crushed with liquid nitrogen. The macerated hyphal material was resuspended in 1:1 volumes of extraction buffer (25 mM Tris–HCl [pH 7.5], 10 mM MgCl2,150 mM NaCl, 1 mM EDTA, 0.01% NP‐40, 2% glycerol, 1 mM Pefabloc [Sigma], 1 mM protein inhibitor cocktail [Sigma]), and the resulting crude lysates were centrifuged at 3,500 rpm for 9 min at 4°C. Cleared lysates were then transferred to new tubes and the total protein concentration was quantified using the Bradford assay. Fifty micrograms of cleared lysate were boiled before SDS‐PAGE separation. Membranes were probed with the anti‐Ras, clone RAS10 mouse monoclonal primary antibody (1:2,000 dilution, EMD Millipore) and followed by the secondary antibody, a horseradish peroxidase‐conjugated goat antimouse IgG2a (1:2,000 dilution, Abcam). Blots were imaged using a Bio‐Rad ChemiDoc XRS HQ System and QuantityOne software (v4.6.5, Bio‐Rad). The assay was performed in biologic triplicate for each strain.
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