Proteins were resolved on 15% SDS-PAGE and stained with Coomassie blue or are transferred to nitrocellulose membrane (Protran, GE Healthcare). Western blotting was performed according to standard procedure with antibodies against Arabidopsis histone variants (5 (link),22 (link)) diluted at 1 μg/ml. The H3 specific antibody (Abcam 1791) was used at 1:5000 dilution. Rat anti-HA antibody (Roche 3F10) was used at 1:2000 dilution. Secondary antibodies were goat anti-rabbit IgG (BioRad) and goat-anti rat IgG (Sigma) at 1:10 000 dilution. Blots were developed with enhanced chemiluminiscence kit (Thermo Fisher Scientific) and signals were recorded by using ChemiDoc instrument (BioRad). Signal quantifications were done with ChemiDoc software by using volume tool.
Enhanced chemiluminiscence kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Enhanced Chemiluminescence Kit is a laboratory reagent designed for the detection and quantification of proteins in Western blot analysis. The kit provides a sensitive and reliable method for the visualization of target proteins by utilizing a chemiluminescent substrate that emits light upon the catalytic action of a horseradish peroxidase (HRP) label.
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Lab products found in correlation
2 protocols using enhanced chemiluminiscence kit
1
Quantitative Immunoblotting of Histone Variants
2
Assessing Nrf2 and Cu-Zn SOD in Testicular Tissue
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Protein was extracted from the nucleus to test the Nrf2 level in nucleus, and whole-cell levels of Cu-Zn SOD were assessed. Testicular tissues were homogenized and fractionated on 10% SDS-PAGE (w/v) gels, and proteins were transferred to a nitrocellulose membrane. Membranes were blocked in 5% non-fat dried milk (w/v) for 1 h, and incubated overnight at 4°C with anti-Cu-Zn SOD (1:1000) and anti-Nrf2 (1:500) antibodies (both Abcam, MA, USA). After unbound antibodies were removed with Tris-buffered saline (pH 7.2) containing 0.05% Tween 20, membranes were incubated at room temperature with secondary antibody for 2 h. Antigenantibody complexes were visualized with an enhanced chemiluminiscence kit (Thermo Scientific, New York, USA). Quantitative densitometry was performed on identified bands using a computerbased measurement system (Zhao et al., 2011) . Histon3 and β-actin were employed as loading controls for the nuclear and cytoplasmic protein, respectively.
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