To evaluate protein expression, whole-cell lysates were prepared, and western blotting was performed. Lysate aliquots containing 30-µg protein were separated by SDS-PAGE gel electrophoresis and transferred onto a Polyvinylidene difluoride membrane (BioRad). The membrane was blocked with 5% skimmed milk powder (Sigma) for 1 h at room temperature, washed with T-TBST and reacted with primary antibody [anti-DNMT1, anti-DNMT3A, anti-DNMT3B, anti-YAP1, anti-ERα and anti-ERβ, anti-actin (all antibodies from Abcam, Cambridge, UK)] at 1:1000 dilution overnight at 4 °C. The specific HRP-labeled secondary antibodies (Abcam) were then reacted at 1:4000 dilutions for 1 h at room temperature. Chemiluminescence was detected using Enhanced Chemiluminescence western blotting detection reagent (BioRad). Actin was used as loading control.
Anti dnmt3a
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-DNMT3A is a protein-specific antibody that recognizes DNA methyltransferase 3A (DNMT3A), an enzyme responsible for de novo DNA methylation. It can be used for various applications such as Western blotting, immunohistochemistry, and immunocytochemistry to detect and study the DNMT3A protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti dnmt3b, by Abcam (19 mentions)
Anti dnmt1, by Abcam (13 mentions)
Anti actin, by Abcam (2 mentions)
Anti p300, by Santa Cruz Biotechnology (2 mentions)
Anti g9a, by Abcam (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Anti dnmt1, by Abnova (2 mentions)
Anti tet2, by Abcam (2 mentions)
Anti dnmt1, by Santa Cruz Biotechnology (2 mentions)
Anti ki67, by Abcam (2 mentions)
Hybond, by Cytiva (2 mentions)
Anti α tubulin, by Abcam (2 mentions)
Enhanced chemiluminescence western blotting detection reagent, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Anti yap1, by Abcam (1 mentions)
Skimmed milk powder, by Merck Group (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti cd68, by Thermo Fisher Scientific (1 mentions)
38 protocols using anti dnmt3a
1
Quantifying Protein Expression via Western Blot
2
Immunoblot Analysis of Cell and Tissue Proteins
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The immunoblot analysis was performed as described previously 35 (link). Briefly, tissues and cells were lysed in 1×RIPA buffer (Sigma-Aldrich, #R0278). Equal amounts of protein in each sample were loaded into a 10% SDS-PAGE gel. After transferring to a membrane and blocking with 5% milk, the proteins were probed with the following primary antibodies: anti-CtBP1 (BD Biosciences, San Jose, CA, USA, #612042), anti-CtBP2 (BD Biosciences, #612044), anti-CD31 (ThermoFisher Scientific, #PA5-16301), anti-CD55 (ThermoFisher Scientific, #PA5-82005), anti-CD68 (ThermoFisher Scientific, #MA5-13324), anti-GAPDH (Santa Cruz Biotechnology, Dallas, Texas, USA, #sc-365062), anti-Caspase-1 (Santa Cruz Biotechnology, #sc-56036), anti-Flag (Sigma-Aldrich, #SAB4200071), anti-Myc (Abcam, Cambridge, MA, USA, #ab9106), anti-p300 (Santa Cruz Biotechnology, #sc-585), anti-c-Jun (Sigma-Aldrich, #SAB4501606), anti-c-FOS (Sigma-Aldrich, #F7799), anti-p50 (ThermoFisher Scientific, #PA1-30409), anti-p65 (ThermoFisher Scientific, #14-6731-81), anti-IRF2 (Abcam, #ab3388), anti-STAT4 (Abcam, #ab68156), anti-NLRP3 (Abcam, #ab210491), anti-Il-1β (Abcam, #ab2105), anti-DNMT1 (Abcam, #ab13537), and anti-DNMT3A (Abcam, #ab2850). After probing with secondary antibodies, protein band signals were detected using a PierceTM ECL western blotting substrate (ThermoFisher Scientific, #32106).
3
Comprehensive ChIP Assay of Epigenetic Markers
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ChIP assay was performed with EZ-Magna ChIP A and EZ-Magna ChIP G Kits (Milliore) according to the manufacturer’s protocol. ChIP grade antibodies were as follows: anti- H3K27me3, anti- H3K9me2, anti-EZH2, normal rabbit IgG, and normal mouse IgG (Milliore); anti-G9a, anti-DnmT1, anti-DnmT3a, and anti-DnmT3b (Abcam). Immunoprecipitated DNA was analyzed by real-time PCR normalized with the input DNA. The sequences of the primers in reference to the TSS regions of the HOXA1 gene are listed in Additional file 5 : Table S4. The primer amplification efficiency of HOXA1 was detected using DNA template gradient dilution method and the amplification efficiency was 99.2%, approaching 100% (Additional file 2 : Figure S1C).
4
Protein Extraction and Western Blot Analysis
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Cell pellets were lysed using NP-40 lysis buffer (150 mM NaCl, 50 mM Tris at pH 8.0, 1%NP-40 supplemented with phosphatase inhibitor cocktails 2 and 3 [Sigma] and protease inhibitor mix [Sigma]). Pellets were vigorously vortexed and incubated for 30 min at 4°C while rotating. Lysates were then quantified using the BCA kit (Thermo Scientific) according to the manufacturer's protocol. Protein sample buffer (3% SDS, 5% β-mercaptoethanol, 10% glycerol, 62 mM Tris at pH 8.0) was added, and samples were loaded on SDS–polyacrylamide gels for electrophoresis. Proteins were transferred to nitrocellulose membranes, blocked with 3% milk in TBST (0.05% Tween-20 in TBS), and incubated with primary antibodies as follows: anti-GAPDH (Millipore), anti-p53 (Biosystems), anti-Dnmt1 (Abnova), anti-Dnmt3a (Abcam), anti Dnmt3b (Abcam), anti TET2 (Abcam), and anti TET1 (GeneTex). Membranes were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated anti-IgG antibodies. Proteins were visualized using the Enhanced Chemo-Luminescence (ECL) detection kit (Amersham).
5
Quantification of DNA Methyltransferases
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The proteins were extracted using the M-PER Mammalian Protein Extraction Reagent (Thermo Scientific). For each sample, 10 μg of total proteins was resolved by SDS-PAGE (10%) and transferred to nitrocellulose membranes. The membranes were incubated with the following primary antibodies overnight at 4°C: anti-DNMT1 (#sc-10219; Santa Cruz Biotechnology), anti-DNMT3A (#ab71424; Abcam), anti-DNMT3B (#ab119282; Abcam), and anti-Albumin (#ab10241; Abcam). The DNMT and ALB antibodies were used at dilutions of 1/500 and 1/1,000, respectively. A 1/1,500 dilution of the anti-β-Tubulin antibody (#T4026; Sigma) was used as a loading control. The antigen-antibody complexes were visualized by chemiluminescence using the ECL Plus western blotting detection system (GE Healthcare) and scanned with the Fujifilm LAS-3000 imaging system (Fujifilm).
6
Western Blot Analysis of Cell Cycle Regulators
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Western blots were performed using anti-DNMT3A (Abcam, Cambridge, UK) and mouse anti-cyclinD1, anti-CDK4, anti-CDK6, anti-p18INK4C, and anti-CDKN1B which were purchased as part of a Cell Cycle Regulation Sampler Kit (Cell Signaling Technology). Mouse anti-β-actin was obtained from Sigma-Aldrich. Protein detection was performed with Super Signal Chemiluminescence Substrate (Pierce, USA).
7
Immunoblot Analysis of Mitochondrial Proteins
8
Protein Expression Analysis in Cells
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Total cellular protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology) on ice. The total protein concentration was measured with a BCA protein assay kit (Takara Biotechnology Co., Ltd.). A total of 30 µg/lane protein was separated by SDS-PAGE on 10% gels and transferred onto PDVF membranes (MilliporeSigma) that were then blocked with 5% BSA for 2 h at room temperature. The following antibodies were added and incubated at 4˚C overnight: Anti-HOXC13 (1:1,000; cat. no. ab168368; Abcam), anti-E-cadherin (1:1,000; cat. no. ab40772; Abcam), anti-N-cadherin (1:1,000; cat. no. ab76011; Abcam), anti-Vimentin (1:1,000; cat. no. ab92547; Abcam), anti-hexokinase II (HK2; 1:1,000; cat. no. ab209847; Abcam), anti-pyruvate kinase M2 (PKM2; 1:1,000; cat. no. ab85555; Abcam), anti-DNMT3A (1:1,000; cat. no. ab188470; Abcam) and anti-GAPDH (1:1,000; cat. no. ab8245; Abcam). Horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5,000; cat. no. ab288151; Abcam) or anti-mouse IgG antibody (1:2,000; cat. no. ab6728; Abcam) was added and incubated at 4˚C overnight. Protein bands were visualized using an enhanced chemiluminescence (ECL)-Plus kit (Thermo Fisher Scientific, Inc.). Blots were analyzed using ImageJ software (1.42q; National Institutes of Health).
9
Chromatin Immunoprecipitation and Analysis
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The indicated cells (4 × 106) in a 100‐mm culture dish were treated with 1% final concentration of formaldehyde to cross‐link proteins to DNA, and the reaction was stopped by the addition of glycine. The cell lysates were sonicated to shear the DNA to fragments of 300–1,000 bp. Chromatin supernatants were incubated with anti‐5mc (#15200081; Diagenode), anti‐DNMT1 (#ab87656; Abcam), anti‐DNMT3A (#ab2850; Abcam), anti‐DNMT3B (#ab13604; Abcam), anti‐H3K27me3 (#9733; Cell Signaling Technology), anti‐H3K9me3 (#9753; Cell Signaling Technology), anti‐H3K4me3 (#9751; Cell Signaling Technology), anti‐SALL2 (#A303‐208A; Bethyl), anti‐p300 (#ab14984; Abcam), anti‐RNA polymerase II (#05‐623; Millipore), or anti‐immunoglobulin G antibody (#I8765; Sigma‐Aldrich) overnight at 4°C with rotation. After reversing the cross‐linking of protein/DNA complexes to free DNA, PCR was performed using primers listed in Appendix Table S9 .
10
Protein Expression Analysis in HCC
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Protein lysates from HCC cells were subjected to Western blotting analysis by using anti-EZH2 (Abcam), anti-DNMT1 (Abcam), anti-DNMT3A (Abcam), anti-DNMT3B (Abcam), and anti-CCNG1 (Abcam) according to standard protocols as previously described [24 ,30 (link)]. Formaldehyde-fixed, paraffin-embedded sections were subjected to H&E staining and immunohistochemistry by using anti-Ki67 (Abcam) following the routine protocols.
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