Revision 1

#88548Store at -20C

1 Kit

(3 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
DNMT1 (D63A6) XP® Rabbit mAb 5032 20 µl 200 kDa Rabbit IgG
DNMT3A (E9P2F) Rabbit mAb 49768 20 µl 85, 95, 130 kDa Rabbit IgG
DNMT3B (E8A8A) XP® Rabbit mAb 57868 20 µl 96 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The DNA Methylation Antibody Sampler Kit provides an economical means of detecting DNA methyltransferase enzymes. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Storage

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 antibodies.

Background

Methylation of DNA at cytosine residues in mammalian cells is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting and development (1,2). Three families of mammalian DNA methyltransferases have been identified: DNMT1, DNMT2, and DNMT3 (1,2). DNMT1 is constitutively expressed in proliferating cells and functions as a maintenance methyltransferase, transferring proper methylation patterns to newly synthesized DNA during replication. DNMT3A and DNMT3B are strongly expressed in embryonic stem cells with reduced expression in adult somatic tissues. DNMT3A and DNMT3B function as de novo methyltransferases that methylate previously unmethylated regions of DNA. DNMT2 is expressed at low levels in adult somatic tissues and its inactivation affects neither de novo nor maintenance DNA methylation. DNMT1, DNMT3A, and DNMT3B together form a protein complex that interacts with histone deacetylases (HDAC1, HDAC2, Sin3A), transcriptional repressor proteins (RB, TAZ-1), and heterochromatin proteins (HP1, SUV39H1) to maintain proper levels of DNA methylation and facilitate gene silencing (3-8). Improper DNA methylation contributes to diseased states such as cancer (1,2). Hypermethylation of promoter CpG islands within tumor suppressor genes correlates with gene silencing and the development of cancer. In addition, hypomethylation of bulk genomic DNA correlates with and may contribute to the onset of cancer. DNMT1, DNMT3A, and DNMT3B are overexpressed in many cancers, including acute and chronic myelogenous leukemias, in addition to colon, breast, and stomach carcinomas (9-12).

  1. Hermann, A. et al. (2004) Cell. Mol. Life Sci. 61, 2571-87.
  2. Turek-Plewa, J. and Jagodziński, P.P. (2005) Cell. Mol. Biol. Lett. 10, 631-47.
  3. Kim, G.D. et al. (2002) EMBO J. 21, 4183-95.
  4. Fuks, F. et al. (2001) EMBO J. 20, 2536-44.
  5. Geiman, T.M. et al. (2004) Biochem. Biophys. Res. Commun. 318, 544-55.
  6. Rountree, M.R. et al. (2000) Nat. Genet. 25, 269-77.
  7. Pradhan, S. and Kim, G.D. (2002) EMBO J. 21, 779-88.
  8. Fuks, F. et al. (2003) Nucleic Acids Res. 31, 2305-12.
  9. Mizuno, S. et al. (2001) Blood 97, 1172-9.
  10. Robertson, K.D. et al. (1999) Nucleic Acids Res. 27, 2291-8.
  11. Xie, S. et al. (1999) Gene 236, 87-95.
  12. Kanai, Y. et al. (2001) Int. J. Cancer 91, 205-12.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #88548

    DNA Methylation Antibody Sampler Kit

    DNA Methylation Antibody Sampler Kit: Image 1 Expand Image
    Western blot analysis of extracts from various cell lines using DNMT3A (E9P2F) Rabbit mAb.
    DNA Methylation Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from 293 and F9 cells using DNMT1 (D63A6) XP® Rabbit mAb.
    DNA Methylation Antibody Sampler Kit: Image 3 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ovarian serous adenocarcinoma using DNMT1 (D63A6) XP® Rabbit mAb.
    DNA Methylation Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using DNMT3B (E8A8A) XP® Rabbit mAb.
    DNA Methylation Antibody Sampler Kit: Image 5 Expand Image
    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.
    DNA Methylation Antibody Sampler Kit: Image 6 Expand Image
    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).
    DNA Methylation Antibody Sampler Kit: Image 7 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    DNA Methylation Antibody Sampler Kit: Image 8 Expand Image
    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 (E9P2F) Rabbit mAb. Western blot analysis was performed using DNMT3A (E9P2F) Rabbit mAb.
    DNA Methylation Antibody Sampler Kit: Image 9 Expand Image
    Confocal immunofluorescent analysis of COS cells using DNMT1 (D63A6) XP® Rabbit mAb (green). Actin filaments were labeled using DY-554 phalloidin (red).
    DNA Methylation Antibody Sampler Kit: Image 10 Expand Image
    Immunohistochemical analysis of paraffin-embedded human soft tissue squamous cell carcinoma using DNMT1 (D63A6) XP® Rabbit mAb.
    DNA Methylation Antibody Sampler Kit: Image 11 Expand Image
    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.
    DNA Methylation Antibody Sampler Kit: Image 12 Expand Image
    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).
    DNA Methylation Antibody Sampler Kit: Image 13 Expand Image
    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).
    DNA Methylation Antibody Sampler Kit: Image 14 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either DNMT3A (E9P2F) Rabbit mAb or DNMT3A (D2H4B) Rabbit mAb #32578, using SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using DNA Library Prep Kit for Illumina #56795. The figures show binding across HUWE1, a known target gene of DNMT3A (see additional figures containing ChIP-qPCR data).
    DNA Methylation Antibody Sampler Kit: Image 15 Expand Image
    Flow cytometric analysis of COS7 cells using DNMT1 (D63A6) XP® Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L) F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    DNA Methylation Antibody Sampler Kit: Image 16 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal human thymus using DNMT1 (D63A6) XP® Rabbit mAb.
    DNA Methylation Antibody Sampler Kit: Image 17 Expand Image
    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).
    DNA Methylation Antibody Sampler Kit: Image 18 Expand Image
    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.
    DNA Methylation Antibody Sampler Kit: Image 19 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and either DNMT3A (E9P2F) Rabbit mAb #49768 or DNMT3A (D2H4B) Rabbit mAb #32578, using SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using DNA Library Prep Kit for Illumina #56795. The figures show binding across HUWE1 (upper) and ARHGEF2 (lower), known target genes of DNMT3A (see additional figures containing ChIP-qPCR data).
    DNA Methylation Antibody Sampler Kit: Image 20 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal human testis using DNMT1 (D63A6) XP® Rabbit mAb.
    DNA Methylation Antibody Sampler Kit: Image 21 Expand Image
    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.
    DNA Methylation Antibody Sampler Kit: Image 22 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells and eitherDNMT3A (E9P2F) 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.
    DNA Methylation Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal human lymph node using DNMT1 (D63A6) XP® Rabbit mAb.
    DNA Methylation Antibody Sampler Kit: Image 24 Expand Image
    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.
    DNA Methylation Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal human placenta using DNMT1 (D63A6) XP® Rabbit mAb (left) or a DNMT1 Rabbit mAb (right). These two antibodies detect unique, non-overlapping epitopes on human DNMT1. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.
    DNA Methylation Antibody Sampler Kit: Image 26 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using DNMT1 (D63A6) XP® Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right). 
    DNA Methylation Antibody Sampler Kit: Image 27 Expand Image
    Immunohistochemical analysis of paraffin-embedded HCT 116 cell pellet, untreated (left) or Doxycycline-treated (10 ng/mL, 72 hr, right), using DNMT1 (D63A6) XP® Rabbit mAb.
    DNA Methylation Antibody Sampler Kit: Image 28 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from HCT116 wild type cells (left panel) and HCT116 DNMT3B/DNMT1 double knockout cells (right panel), both treated with doxorubicin (1 uM for 24 hrs), and either DNMT1 (D63A6) XP® Rabbit mAb #5032 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 human POMC promoter primers and human Stat2 promoter primers. 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.
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.