Tumor tissues or cells were homogenized in lysis buffer (50 mM Tris-HCL, pH 7.5, 150 mM NaCl, 1% NP40) with Halt™ Protease Inhibitor Cocktail (1861278, Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration in tissues or cell lysates was determined with a protein assay kit (Bio-Rad) using bovine serum albumin as a standard. Aliquots of protein lysate (30 μg) were separated by SDS-PAGE, and Western blots were performed using standard procedures. Blots were developed using the Amersham ECL Western Blotting Detection Reagents kit (GE Healthcare UK Ltd, Buckinghamshire, UK). Primary antibodies used in this study include LC3A (#4599; Cell Signaling Technology, Inc., Danvers, MA), LC3B (#3868; Cell Signaling Technology), Cleaved Caspase-3 (#9664; Cell Signaling Technology), PARP (#5625; Cell Signaling Technology), PCNA (sc-56, Santa Cruz Biotechnology), and GAPDH (#5174; Cell Signaling Technology).
Amersham ecl western blotting detection reagents kit
Manufactured by GE Healthcare
Sourced in United Kingdom
The Amersham ECL Western Blotting Detection Reagents kit is a laboratory equipment product designed for the detection of proteins in Western blotting applications. The kit contains reagents that enable the visualization of target proteins on a membrane using a chemiluminescent detection method.
Automatically generated - may contain errors
Lab products found in correlation
Amersham ecl western blotting, by Cytiva (4 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions)
Hybond p, by Cytiva (1 mentions)
Hybond, by Cytiva (1 mentions)
4 protocols using amersham ecl western blotting detection reagents kit
1
Western Blot Analysis of Apoptosis and Proliferation Markers
2
Western Blot Analysis of LC3 Proteins
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Tumor tissues or cells were homogenized in lysis buffer (50 mmol/L Tris-HCL, pH 7.5, 150 mmol/L NaCl, 1% NP40) with Halt Protease Inhibitor Cocktail (1861278, Thermo Fisher Scientific). Protein concentrations in tissues or cell lysates were determined with a protein assay kit (Bio-Rad), using bovine serum albumin as a standard. Aliquots of protein lysate (30 mg) were separated by SDS-PAGE, and Western blot analyses were performed using standard procedures. Blots were developed using an Amersham ECL Western Blotting Detection Reagents kit (GE Healthcare UK), using primary antibodies LC3A (#4599), LC3B (#3868), and GAPDH (#5174; all from Cell Signaling Technology).
3
Histone Extraction for Western Blot
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Histones were enriched from cell lysates following the AbCam protocol “Histone extraction protocol for western blot” (reference http://www.abcam.com/protocols/histone-extraction-protocol-for-western-blot ).
Following incubation the tubes were briefly vortexed, then centrifuged for 10 min at 6500 x g and 4 ° C. The histone containing supernatant (~400 ul) was transferred to new tubes and 100 ul of Trichloroacetic acid was added. The tubes were vortexed briefly and incubated on ice for 1.5 h followed by centrifugation for 15 min at 16,000 x g and 4 ° C. The supernatant was aspirated and the pelleted protein was washed twice with -20 °C acetone and allowed to dry with the lid open at room temperature for 1.5 h. Pelleted histones were either stored at -20 ° C or immediately subject to SDS-PAGE and Western blot. A target of 5 μg of protein was used for Western blotting. Membranes were incubated for 1 h at ambient temperature with a 1:1000 dilution of anti-H3K27me3 primary antibody (monoclonal mouse) in PBST + 3 % milk solution. Membranes were washed extensively in PBST and incubated in 1:5000 HRP-conjugated anti-mouse secondary antibody (polyclonal goat) for 1 h at room temperature in PBST + 5 % milk. Membranes were washed extensively with PBST and treated with Amersham ECL Western Blotting Detection Reagents kit (cat:28-392766, GE Healthcare).
Following incubation the tubes were briefly vortexed, then centrifuged for 10 min at 6500 x g and 4 ° C. The histone containing supernatant (~400 ul) was transferred to new tubes and 100 ul of Trichloroacetic acid was added. The tubes were vortexed briefly and incubated on ice for 1.5 h followed by centrifugation for 15 min at 16,000 x g and 4 ° C. The supernatant was aspirated and the pelleted protein was washed twice with -20 °C acetone and allowed to dry with the lid open at room temperature for 1.5 h. Pelleted histones were either stored at -20 ° C or immediately subject to SDS-PAGE and Western blot. A target of 5 μg of protein was used for Western blotting. Membranes were incubated for 1 h at ambient temperature with a 1:1000 dilution of anti-H3K27me3 primary antibody (monoclonal mouse) in PBST + 3 % milk solution. Membranes were washed extensively in PBST and incubated in 1:5000 HRP-conjugated anti-mouse secondary antibody (polyclonal goat) for 1 h at room temperature in PBST + 5 % milk. Membranes were washed extensively with PBST and treated with Amersham ECL Western Blotting Detection Reagents kit (cat:28-392766, GE Healthcare).
4
DNMT3A Expression in Tammar Wallaby
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Protein was extracted from day 64 pp tammar PY testis (n = 4 pooled), day 80 pp ovary (n = 5 pooled), and day 23 of gestation fetus (n = 2 pooled) by homogenization in RIPA buffer (50 mM Tris base, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate). Fifty micrograms of each sample were electrophoresed in a 7.5% SDS-PAGE gel and then transferred to a PVDF membrane (Amersham Hybond-P). The membrane was blocked in 5% skim milk dissolved in TBST (TBS, 50 mM Tris HCl, 150 mM NaCl, pH 7.5 with 0.1% Tween 20) at 4 °C overnight and then incubated at room temperature with the anti-DNMT3A antibody (ab13888, 2 µg/ml) for 2 h, followed by a 1 h incubation with a HRP-conjugated goat anti-mouse secondary antibody diluted in TBS with 5% skim milk and 0.1% Triton X-100. Chemiluminescence using the Amersham ECL Western Blotting Detection Reagents kit (GE Healthcare Life Sciences) was used to detect bound antibody.
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