Western blot analysis was performed using standard techniques. Briefly, transiently transfected leukemia cells were harvested and lysed by RIPA buffer (Thermo Scientific, Waltham, MA, USA). Proteins (40 μg) from the lysate were fractionated by electrophoresis through polyacrylamide gels (Bio-Rad, Richmond, CA, USA) and transferred to PVDF membranes. Blots were incubated with primary antibodies over night at 4 °C and incubated with second HRP-conjugated antibody for 1 h. Signals were measured by chemiluminescence reagents (Bio-Rad) with imaging system (Bio-Rad). The following antibody was used: HOXB3 (ab82945, Abcam, Cambridge, MA, USA); CDCA3 (ab166902, Abcam); DNMT3B (ab176166, Abcam). As necessary, blots were stripped and reprobed with β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, US) as an endogenous control.
Ab176166
Manufactured by Abcam
Sourced in United States
Ab176166 is a lab equipment product from Abcam. It is a core function device for use in scientific research.
Automatically generated - may contain errors
Lab products found in correlation
Ab188470, by Abcam (2 mentions)
Ab87654, by Abcam (2 mentions)
Ab1791, by Abcam (2 mentions)
Mab 2118l, by Cell Signaling Technology (2 mentions)
Ab13537, by Abcam (2 mentions)
Ab13888, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Polyacrylamide gel, by Bio-Rad (1 mentions)
Chemiluminescence reagent, by Bio-Rad (1 mentions)
Ab166902, by Abcam (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Chromatin immunoprecipitation chip assay kit, by Merck Group (1 mentions)
Chip kit, by Merck Group (1 mentions)
6 protocols using ab176166
1
Western Blot Analysis of Leukemia Cells
2
DNMT3B Binding to Pre-miR-375 Promoter
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The binding activity of DNMT3B in pre-miR-375 promoter was examined by Chromatin Immunoprecipitation (ChIP) Assay Kit (Merck-Millipore, Billerica, MA, USA) according to the manufacturer’s instruction. Briefly, treated and untreated leukemic cells were cross-linked with 1% formaldehyde for 10 min. Chromatin from nuclear extracts was sonicated to generate 200–1000 bp DNA fragments. DNA-protein complexes were immunoprecipitated with 5 μg of specific anti-DNMT3B (ab176166, Abcam). DNA was purified after the DNA-protein cross-link was reversed by heating at 65 °C for 4 h. Standard PCR reactions were performed by primer pairs (Additional file 2 : Table S2). The immunoprecipitated DNA was analyzed by qRT-PCR and the amount of precipitated DNA was calculated as the percentage of the input sample.
3
Quantifying Epigenetic Regulators in Nuclei
4
Quantifying Epigenetic Regulators in Nuclei
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Cells were collected and washed twice in PBS before being resuspended in cell lysis buffer (25 mM HEPES pH7.6, 5 mM MgCl2, 25 mM KCl, 0.05 mM EDTA, 10% Glycerol, 0.1% IGEPAL, 1× Roche protease inhibitor, 1 mM DTT). The cell lysate was spun for 5 minutes at 1,500 rpm to pellet the nuclei. Nuclei were washed once to remove cell debris (10 mM HEPES pH7.6, 3 mM MgCl2, 100 mM KCl, 0.01 mM EDTA, 10% glycerol, 1× Roche protease inhibitor, 1 mM DTT). Nuclei were spun down at 3,000 g for 5 minutes. Nuclei were then resuspended in 150 μl RIPA buffer and vortexed for 20 minutes at 4°C. This mixture was spun at 12,000 rpm for 15 minutes and the supernatant was collected. 20 μl of 4× LDS buffer was then added to 60 μl of supernatant and denatured at 72°C for 10 minutes. Western blots were performed with anti-Dnmt1 (1:1,000 dilution, Abcam ab87654 lot: GR3194562–7), anti-Dnmt3a (1:2,000, Abcam ab188470 lot: GR224165–2), anti-Dnmt3b (1:500, Abcam ab176166 lot: GR3199224–3), anti-histone H3 (Abcam ab1791 lot: GR293197–1) and anti-GAPDH (Cell Signaling Technology mAb#2118L lot: 10) antibodies and imaged using HRP chemiluminescence.
5
Investigating LncRNA-DNA Methyltransferase Interactions
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The cells were collected using cell scrape, washed twice with pre-cooled PBS, and split with the addition of 100 μl of lysis buffer containing proteinase inhibitors and ribonuclease inhibitors on ice for 30 min. After centrifugation at 120,000 g at 4°C for 30 min, the supernatant was subsequently transferred into a centrifuge tube. A small amount of supernatant was taken as the input positive control. Next, 1 μg corresponding antibodies: anti-Dnmt1 (ab13537, mouse antibody), anti-Dnmt3a (ab13888, mouse antibody), anti-Dnmt3b (ab176166, mouse antibody; all from Abcam Inc.) and 10–50 μl protein A/G-beads were added to the remaining supernatant, and incubated overnight at 4°C. After immunoprecipitation, centrifugation at 3000 g at 4°C for 5 min was conducted, the supernatant was discarded. The protein A/G-beads were then washed with 1 ml lysis buffer, and deposited 3–4 times. After each period of washing, the sample was centrifuged with 1000 g at 4°C for 1 min. Finally, 2 × SDS buffer (15 μl) was added, and heated in boiled water for 10 min. Next, the relative RNA was obtained by isolation and purification from the precipitation. The binding effect of LncRNA and DNA methyltransferase was identified using the specific primers of LINC01419 and RT-qPCR.
6
GSTP1 Gene Promoter-DNA Methyltransferase Binding
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The binding of the GSTP1 gene promoter to the DNA methyltransferase was verified in connection with the instructions of the chromatin immunoprecipitation (ChIP) kit (Millipore, Billerica, MA, USA). Next, 1% formalin was employed to fix the cells for 10 min in order to facilitate DNA and protein cross-linking. Next, DNA was randomly fragmented to 200–800 base pairs (bp) by ultrasonic, and immunoprecipitated with the target protein specific antibodies: anti-Dnmt1 (ab13537, mouse antibody), anti-Dnmt3a (ab13888, mouse antibody), anti-Dnmt3b (ab176166, mouse antibody; all from Abcam Inc.) and IgG in the control group. Finally, 100 µl H2O was used to purify and elute 2.5 µl ChIP DNA which was obtained for RT-qPCR purposes. The binding of the GSTP1 gene promoter and DNA methyltransferase was detected using the GSTP1 primers.
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