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Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro233 of human DNMT3A protein.
Product Usage Information
For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits. The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652. The CUT&Tag dilution was determined using CUT&Tag Assay Kit #77552.
| Application | Dilution |
|---|---|
| Western Blotting | 1:1000 |
| Immunoprecipitation | 1:50 |
| Immunofluorescence (Immunocytochemistry) | 1:800 - 1:1600 |
| Flow Cytometry (Fixed/Permeabilized) | 1:1600 |
| Chromatin IP | 1:50 |
| Chromatin IP-seq | 1:50 |
| CUT&RUN | 1:50 |
| CUT&Tag | 1:50 |
Specificity/Sensitivity
Species Reactivity:
Human
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
参考图片
CUT&Tag was performed with NCCIT cells and DNMT3A (D2H4B) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figures show binding across HUWE1 (upper) and TFRC (lower), which are known target genes of DNMT3A (see our ChIP-qPCR figure).
Western blot analysis of extracts from NCCIT, NTERA-2 cl.D1, and HCT 116 cells using DNMT3A (D2H4B) Rabbit mAb. This antibody detect multiple isoforms of DNMT3A, including isoforms 1 and 2.
Immunoprecipitation of DNMT3A from NCCIT cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is DNMT3A (D2H4B) Rabbit mAb. Western blot analysis was performed using DNMT3A (E9P2F) Rabbit mAb #49768.
Confocal immunofluorescent analysis of NCCIT (left, high-expressing) and 293T (right, low-expressing) cells using DNMT3A (D2H4B) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). According to WB analysis, 293T cells preferentially express the larger DNMT3A isoform 1, while NCCIT cells also express high levels of two smaller isoforms, including isoform 2.
Flow cytometric analysis of PC-3 cells (blue) and NCCIT cells (green) using DNMT3A (D2H4B) Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either DNMT3A (E9P2F) Rabbit mAb #49768 or DNMT3A (D2H4B) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across HUWE1, a known target gene of DNMT3A (see additional figures containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either DNMT3A (E9P2F) Rabbit mAb #49768 or DNMT3A (D2H4B) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across HUWE1 (upper) and ARHGEF2 (lower), known target genes of DNMT3A (see additional figures containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either DNMT3A (D2H4B) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human HUWE1 Intron 26 Control Primers #33770, human ARHGEF2 intron 13 primers, human SDHAP1 intron 10 primers, and SimpleChIP® Human GAPDH Exon 1 Primers #5516. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT&RUN was performed with NCCIT cells and DNMT3A (D2H4B) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across HUWE1, a known target gene of DNMT3A (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with NCCIT cells and DNMT3A (D2H4B) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across TFRC (upper) and HUWE1 (lower), a known target gene of DNMT3A (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with NCCIT cells and either DNMT3A (D2H4B) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human HUWE1 Intron 26 Control Primers #33770, human ARHGEF2 intron 13 primers, human SDHAP1 intron 10 primers, and SimpleChIP® Human GAPDH Exon 1 Primers #5516. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT&Tag was performed with NCCIT cells and DNMT3A (D2H4B) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across HUWE1, a known target gene of DNMT3A (see our ChIP-qPCR figure).