Cell fractions were lysed in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA supplemented with anti-protease and anti-phosphatase cocktails (Roche). Mitochondrial fraction was obtained with the help of a kit from Pierce. The protein concentration was determined using the Bio-Rad DC kit, and 70 μg of protein was loaded in linear SDS-PAGE gels. After blotting, nitrocellulose filters were probed with primary antibodies against CD47 (clone 2D3; eBioscience), activated caspase-3 (9661; Cell Signaling Technology), PLCγ1 (clone D9H10; Cell Signaling Technology), PLCγ1-Y783 (2821; Cell Signaling Technology), Cox IV (clone 1D6E1A8; Life Technologies), DRP1/DLP1 (clone 8/DLP1; BD Biosciences), and α-tubulin (clone B-5–1–2; Sigma). Immunoreactive proteins were detected using HRP-conjugated secondary antibodies and visualized with the ECL system (Thermo Scientific). Immunoblot images were acquired on a MF-ChemiBIS 4.2 (DNR Bio-Imaging Systems). PLCγ1-Y783 was quantified using Multi Gauge 3.0 software (Fujifilm Life Sciences). The optical density was normalized to the background and was expressed relative to the untreated cells (set at 1.0).
Mf chemibis 4
Manufactured by DNR Bio-Imaging Systems
The MF-ChemiBIS 4.2 is a compact and versatile lab equipment designed for chemiluminescence and fluorescence detection. It features a high-resolution camera and advanced optics for capturing high-quality images of samples. The MF-ChemiBIS 4.2 is suitable for a range of applications in the field of molecular biology and biochemistry.
Automatically generated - may contain errors
Lab products found in correlation
α tubulin clone b 5 1 2, by Merck Group (1 mentions)
Anti protease and anti phosphatase cocktails, by Roche (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Dc kit, by Bio-Rad (1 mentions)
Activated caspase 3, by Cell Signaling Technology (1 mentions)
Multi gauge 3, by Fujifilm (1 mentions)
Gene images alkphos direct labeling and detection system kit, by GE Healthcare (1 mentions)
Cdp star, by Cytiva (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
2 protocols using mf chemibis 4
1
Fractionation and Immunoblotting of Cellular Proteins
2
AIF-Deficient MEF DNA Analysis
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Genomic DNA from WT (Co) and AIF-deficient (AIF-/Y) MEFs (4 days post-treatment with tamoxifen; 4-OHT) was extracted by using a Wizard Genomic DNA purification kit (Promega) and digested with Pci1. The genomic DNA fragments obtained, including those encompassing exons 7 to 13 of Aifm1, were separated on a 0.8% agarose gel and transferred to a hybond membrane. The membrane was baked for 2 h at 80 °C to fix DNA and then hybridized with a probe labeled with a Gene Images AlkPhos direct labeling and detection system kit from GE Healthcare. The probe, specific to the 3'end of AIF genomic DNA, was freshly prepared by PCR amplification of genomic DNA by using sense (intron 12 nucleotides 33070–33098) and reverse (intron 13 nucleotides 33433–33459) primers. The membrane was then washed, and the hybridization pattern was revealed by using CDP star (Amersham). Images were acquired on a MF-ChemiBIS 4.2 (DNR Bio-Imaging Systems).
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