1

5-mC monoclonal antibody 33D3

Description: Monoclonal antibody raised in mouse against 5-mC (5-methylcytosine) conjugated to ovalbumine.

Applications

Suggested dilution Results

MeDIP/MeDIP-seq* 1 - 2 µg per IP Fig 1-2

ELISA 1:100 Fig 3

Dot blotting 1:250 Fig 4

Immunofluorescence 1:500 Fig 5

* Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-5 µg per IP.

This antibody has been described in:(1) Natt D, Rubin CJ, Wright D, Johnsson M, Belteky J, Andersson L, Jensen P (2012) Heritable genome-wide variation of gene

expression and promoter methylation between wild and domesticated chickens. BMC Genomics 13: 59.

(2) Gavin DP, Sharma RP, Chase KA, Matrisciano F, Dong E, Guidotti A (2012) Growth Arrest and DNA-Damage-Inducible, Beta (GADD45b)-Mediated DNA Demethylation in Major Psychosis. Neuropsychopharmacology 37: 531-542.

(3) Sharp AJ, Stathaki E, Migliavacca E, Brahmachary M, Montgomery SB, Dupre Y, Antonarakis SE (2011) DNA methylation profiles of human active and inactive X chromosomes. Genome Res 21: 1592-1600.

(4) Katsurano M, Niwa T, Yasui Y, Shigematsu Y, Yamashita S, Takeshima H, Lee MS, Kim YJ, Tanaka T, Ushijima T (2011) Early-stage formation of an epigenetic field defect in a mouse colitis model, and non-essential roles of T- and B-cells in DNA methylation induction. Oncogene 31:342-351.

(5) Zuo T, Liu TM, Lan X, Weng YI, Shen R, Gu F, Huang YW, Liyanarachchi S, Deatherage DE, Hsu PY, Taslim C, Ramaswamy B, Shapiro CL, Lin HJ, Cheng AS, Jin VX, Huang TH (2011) Epigenetic silencing mediated through activated PI3K/AKT signaling in breast cancer. Cancer Res 71: 1752-1762.

(6) Son JS, Chae CS, Hwang JS, Park ZY, Im SH (2011) Enhanced chromatin accessibility and recruitment of JUNB mediate the sustained IL-4 expression in NFAT1 deficient T helper 2 cells. PLoS One 6: e22042.

(7) Sharp AJ, Migliavacca E, Dupre Y, Stathaki E, Sailani MR, Baumer A, Schinzel A, Mackay DJ, Robinson DO, Cobellis G, Cobellis L, Brunner HG, Steiner B, Antonarakis SE (2010) Methylation profiling in individuals with uniparental disomy identifies novel differentially methylated regions on chromosome 15. Genome Res 20: 1271-1278.

(8) Hsu PY, Hsu HK, Singer GA, Yan PS, Rodriguez BA, Liu JC, Weng YI, Deatherage DE, Chen Z, Pereira JS, Lopez R, Russo J, Wang Q, Lamartiniere CA, Nephew KP, Huang TH (2010) Estrogen-mediated epigenetic repression of large chromosomal regions through DNA looping. Genome Res 20: 733-744.

Cat. No. MAb-081-010 (sample), MAb-081-100 (standard), MAb-081-500 (large)Type: Monoclonal MeDIP-grade, MeDIP-seq grade ; IgG1Source: MouseClone: 33D3Lot #: GF-002Size: 10 µg/ 5 µl ;100 µg/ 50 µl ; 500 µg/ 250 µlConcentration: 2 µg/µl

Specificity: predicted to react with 5-mC found in all vertebrate and plant species.

Purity: Monoclonal antibody purified by gel filtration, in PBS containing 0.05% azide.Storage: Store in small aliquots at -80°C. Avoid multiple freeze-thaw cycles.Precautions: This product is for research use only. Not for use in diagnostic or therapeutic procedures.

2

(9) Ruike Y, Imanaka Y, Sato F, Shimizu K, Tsujimoto G (2010) Genome-wide analysis of aberrant methylation in human breast cancer cells using methyl-DNA immunoprecipitation combined with high-throughput sequencing. BMC Genomics 11: 137.

(10) Lewis ZA, Honda S, Khlafallah TK, Jeffress JK, Freitag M, Mohn F, Schübeler D, and Selker EU (2009) Relics of repeat-induced point mutation direct heterochromatin formation in Neurospora crassa Genome Res 19: 427–437.

(11) Cohen NM, Dighe V, Landan G, Reynisdóttir S, Palsson A, Mitalipov S, Tanay A (2009) DNA methylation programming and reprogramming in primate embryonic stem cells. Genome Res 19: 2193-2201.

(12) Takeshima H, Yamashita S, Shimazu T, Niwa T, Ushijima T (2009) The presence of RNA polymerase II, active or stalled, predicts epigenetic fate of promoter CpG islands. Genome Res 19: 1974-1982.

(13) Yamashita S, Hosoya K, Gyobu K, Takeshima H, Ushijima T (2009) Development of a novel output value for quantitative assessment in methylated DNA immunoprecipitation-CpG island microarray analysis. DNA Res 16: 275-286.`

Results

Figure 1. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) with the Diagenode 5-mC (clone33D3) antibody

Genomic DNA from E14 ESC cells was prepared and sonicated using a Bioruptor® to generate random fragments (size range 300 to 700 bp). 1 µg of fragmented DNA was ligated to Illumina adapters and the resulting DNA was used for a standard MeDIP-seq assay (using 2µg of the Diagenode monoclonal against 5-mC (Cat. No. MAb-081-010/100/500) per assay). After recovery of the methylated DNA, Illumina sequencing libraries were generated and sequenced on an Illumina Genome Analyzer according to the manufacturer’s instructions. Figure 1A and 1B show Genome browser views of CA simple repeat elements with read distributions specific for 5-mC at 2 gene locations (SigleC15 and Mfsd4). Visual inspection of the peak profiles in a genome browser shows high enrichment of CA simple repeats in affinity-enriched methylated fragments using MeDIP with the Diagenode 5-mC monoclonal (33D3 clone).

1A

1B

3

Figure 4. Dot blot analysis using the Diagenode monoclonal antibody directed against 5-mC

Figure 4A. To demonstrate the specificity of the Diagenode antibody against 5-mC (Cat. No. MAb-081-010/100/500), a Dot blot analysis was performed using “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002). One hundred to 4 ng of the controls were spotted on a membrane (Amersham Hybond-N+). The antibody was used at a dilution of 1:250.

Figure 4B. Dot blot was performed as described above on decreasing amounts of methylated DNA isolated from phage XP12.

4A 4B

Figure 3. ELISA using the Diagenode monoclonal antibody directed against 5-mC

ELISA was performed using Diagenode monoclonal antibody against 5-mC (Cat. No. MAb-081-010/100/500), diluted 1:100. The wells were coated with a serial dilution of the methylated DNA control from the Diagenode «5-hmC, 5-mC & cytosine DNA standard pack » (Cat No. AF-101-0002).

Figure 2. MeDIP results obtained with the Diagenode monoclonal antibody directed against 5-mC

MeDIP (Methylated DNA immunoprecipitation) was performed on fragmented genomic DNA from U2OS cells using the Diagenode monoclonal antibody against 5-mC (Cat. No. MAb-081-010/100/500) and the MagMeDIP Kit (Cat. No. mc-magme-048). The fragmented DNA was spiked with methylated DNA (meDNA) as a positive and unmethylated DNA (unDNA) as a negative control. QPCR was performed with optimized primer sets, included in the kit, specific for the methylated and unmethylated DNA controls, and for a known methylated (TSH2B) and unmethylated (GAPDH) genomic region. An additional internal positive and negative control locus (4994+ and 8804-, respectively) was also tested.

QPCR was performed with optimized primer sets, included in the kit, specific for the methylated and unmethylated DNA controls, and for a known methylated (TSH2B) and unmethylated (GAPDH) genomic region. An additional internal positive and negative control locus (4994+ and 8804-, respectively) was also tested (4994+: forward primer 5’-GGGAATATAAGGAGCGCACA-3’ and reverse primer 5’- TCGGTTAAAACGGTCAGGTC-3’; 8804-: forward primer 5’-CGAGGCGTGAGTTATTCCTG-3’ and reverse primer 5’-CTCTTGTGGCTGAGCTCCTT-3’). Figure 2 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

% o

f IN

PU

T10

-

20

30

40

50

60

70

80

62,1

0,5 0,3

71,6

60,6

0,2

meDNA unDNA GAPDH TSH2B 4994+ 8804-

4

Diagenode sa. BELGIUM | EUROPE

Avenue de l’hôpital,1Tour GIGA, 3rd Floor4000 Liège - BelgiumTel: +32 4 364 20 50Fax: +32 4 364 20 51orders@diagenode.cominfo@diagenode.com

Diagenode Inc. USA | NORTH AMERICA

400 Morris Avenue, Suite 101Denville, NJ 07834 - USATel: +1 862 209-4680Fax: +1 862 209-4681orders.na@diagenode.cominfo.na@diagenode.com

Last update: June 13, 2012

0 50 100 150 200

Time s

250 300 350 400 450-100

-50

0

50

100

150

200

250

RU

Response

Figure 6. Surface plasmon resonance (SPR) analysis of the the Diagenode 5-mC monoclonal antibody, 33D3 clone (MAB-081-100; MAB-081-500).

The synthesized biotin-labeled 5-mC conjugate was immobilized on a CM4 BIAcore sensorchip (GE Healthcare, France), as described below. Briefly, two flowcells were prepared by sequential injections of EDC/NHS, streptavidin, and ethanolamine. One of these flowcells served as negative control (biotinylated spacer without 5-mC), while biotinylated 5-mC conjugate was injected on the other one, to get an immobilization level of 55 response units (RU). All SPR experiments were performed, using HBS-N buffer (10 mM HEPES,150 mM NaCl, pH 7.4), at a flow rate of 5 µl/min. Interaction assays involved injections of 2 different dilutions of the Diagenode 5-mC monoclonal antibody 33D3 clone (Cat. No. MAb-081-010/100/500) (2mg/ml, brown line; 0.37 mg/ml, blue line; final working concentration (100 µg/ml) over the biotinylated 5-mC conjugate and negative control surfaces, followed by a 3 min washing step with HBS-N buffer to allow dissociation of the complexes formed. At the end of each cycle, the streptavidin surface was regenerated by injection of 0.1M citric acid (pH=3).

Sensorgrams shown correspond to on-line subtraction of the negative control to the biotinylated 5-mC conjugate surface signal. Data from sensorgrams that reached binding equilibrium were used for Scatchard analysis. As showen in the figure below the three curves are clearly overlapping to each other and the value of dissociation constant (kd) obtained by global fitting and 1:1 Langmuir model is 22 ± 4.5 nM.

Figure 5. Immunofluorescence results obtained with the Diagenode monoclonal antibody directed against 5-mC

Figure 5A. Human osteosarcoma (U2OS) cells were stained with the Diagenode monoclonal antibody against 5-mC (Cat. No. MAb-081-010/100/500). Cells were fixed with 2.5% PFA in PBS for 30’, permeabilised with 0.5% Triton X-100 for 1 hour and treated with 2N HCl for 1 hour followed by 2 x 5 minutes with 0.1 M borate buffer to depurinate the DNA. After blocking with PBS containing 0.1% TritonX-100 and 1% BSA, the cells were immunofluorescently labeled with the 5-mC antibody diluted 1:500 in blocking solution, followed by a goat anti-mouse antibody conjugated to Alexa488.

Figure 5B: Immunofluorescent staining of an interphase HeLa cell with the Diagenode 5-mC antibody followed by a goat anti-mouse antibody conjugated to FITC (yellow) and with DAPI (blue).

5A

5B