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Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human DNMT3B 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 | 1:100 |
| Immunofluorescence (Immunocytochemistry) | 1:1600 |
| Flow Cytometry (Fixed/Permeabilized) | 1:50 - 1:200 |
| Chromatin IP | 1:50 |
| CUT&RUN | 1:50 |
| CUT&Tag | 1:50 |
Specificity/Sensitivity
Species Reactivity:
Human, Monkey
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.
For a carrier free (BSA and azide free) version of this product see product #84194.
参考图片
CUT&Tag was performed with NCCIT cells and DNMT3B (E8A8A) XP® 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 the HUWE1 gene (upper) and the SMARCA1 gene (lower).
Western blot analysis of extracts from various cell lines using DNMT3B (E8A8A) XP® Rabbit mAb.
Immunoprecipitation of DNMT3B from NCCIT cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is DNMT3B (E8A8A) XP® Rabbit mAb. Western blot analysis was performed using DNMT3B (E8A8A) XP® Rabbit mAb and Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127.
Confocal immunofluorescent analysis of NCCIT cells (left, high-expressing), HCT 116 DNMT3B wild-type cells (middle, low-expressing), and HCT 116 DNMT3B knockout cells (right, negative) using DNMT3B (E8A8A) Rabbit XP® mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Flow cytometric analysis of NCCIT cells (green), HCT116 WT cells (red) or HCT116 DNMT3B KO cells (blue), using DNMT3A (E8A8A) XP® Rabbit mAb (solid lines) or 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 DNMT3B (E8A8A) XP® Rabbit mAb #57868 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 EIF4G1 Intron 8 Primers #29118, human CROCC intron 1 primers, and SimpleChIP® Human GAPDH Promoter Primers #4471. 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 either DNMT3B (E8A8A) XP® Rabbit mAb or DNMT3B (E2Q3Z) Rabbit mAb #72335, 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 HUWE1, a known target gene of DNMT3B (see additional figures containing CUT&RUN-qPCR data).
CUT&RUN was performed with NCCIT cells and either DNMT3B (E8A8A) XP® Rabbit mAb or DNMT3B (E2Q3Z) Rabbit mAb #72335, 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 HUWE1 (upper) and SMARCA1 (lower), known target genes of DNMT3B (see additional figures containing CUT&RUN-qPCR data).
CUT&RUN was performed with NCCIT cells and either DNMT3B (E8A8A) XP® 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 human HUWE1 intron 26 primers, human ARHGEF2 promoter 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 DNMT3B (E8A8A) XP® 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 the HUWE1 gene.