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Superior Performance of New DNA Methylation Recombinant Rabbit Monoclonal Antibodies

Sigma’s recombinant rabbit monoclonal antibodies, anti-5-mC (Clone RM231, SAB5600040) and anti-5-hmC (clone RM236, SAB5600041), are the first of its kind developed against epigenetic targets. Selected from well diversified immune repertoire, these clones have gone through extensive screening and validation,  outperforming their rodent monoclonal counterparts currently available in the market. Recombinant technology ensures the lot to lot consistency.

Excellent Specificity

ELISA shows no cross-reactivity between anti-5-mC and anti-5-hmC rabbit monoclonal antibodies (mAbs).

ELISA of single stranded DNA using Anti-5-mC

ELISA of single stranded unmethylated, 5-mC, and 5-hmC DNA, using anti-5-mC rabbit mAb, or anti-5-hmC rabbit mAb.
The plate was coated with streptavidin and then biotinylated single stranded unmethylated DNA, 5-mC DNA, and 5-hmC DNA. Anti-5-mC rabbit mAb (clone RM231, Product No. SAB5600040), or anti-5-hmC rabbit mAb (clone RM236, Product No. SAB5600041) was used as the primary antibody, and an alkaline phosphatase conjugated anti-rabbit IgG as the secondary antibody.

High Affinity

With larger immune repertoire, rabbit B cells can better recognize foreign epitope with much higher affinity.

Dot blot of double stranded DNA using rabbit monoclonal anti-5-mC or anti-5-hmC antibody

Dot blot of double stranded DNA using rabbit monoclonal anti-5-mC or anti-5-hmC antibody.
The membrane was pre-spotted with 50, 5, and 0.5 ng/dot of double stranded 5-Hydroxymethylcytosine (5-hmC) DNA, 5-Methylcytosine (5-mC) DNA, and unmethylated DNA. Then the pre-spotted membrane was blotted with anti-5-mC (clone RM231, Product No. SAB5600040) or anti-5-hmC (clone RM236, Product No. SAB5600041).


ELISA of single stranded DNA using rabbit monoclonal anti-5-mC antibody

ELISA of single stranded DNA using rabbit monoclonal anti-5-hmC antibody

ELISA of single stranded DNA using rabbit monoclonal anti-5-mC or anti-5-hmC antibody.
The plate was coated with streptavidin and then biotinylated single stranded unmethylated DNA, 5-Methylcytosine (5-mC) DNA, and 5-Hydroxymethylcytosine (5-hmC) DNA. A serial dilution of anti-5-mC (clone RM231, Product No. SAB5600040) or RM236 (clone RM236, Product No. SAB5600041) was used as the primary antibody, and an alkaline phosphatase conjugated anti-rabbit IgG as the secondary antibody.

Low Background with Various Applications

With extensive screening and validation, antibodies are well defined for IHC and other applications. Recombinant technology creates the reproducible high quality antibodies.

ICC staining of HeLa cells using rabbit monoclonal anti-5-mC

Immunocytochemical staining of HeLa cells using rabbit monoclonal anti-5-mC (clone RM231, Product No. SAB5600040) antibody (red). Actin filaments have been labeled with fluorescein phalloidin (green), and nuclei stained with DAPI (blue).

ICC staining of human brain tissue sections, using rabbit monoclonal anti-5-hmC

Immunohistochemical staining of formalin fixed and paraffin embedded human brain tissue sections, using rabbit monoclonal anti-5-hmC (clone RM236, Product No. SAB5600041) antibody.

Superior Performance

With higher sensitivity, Sigma anti-5-mC and anti-5-hmC recombinant rabbit monoclonal antibodies identifies more than 2 times signals of those of competitor antibodies.

Comparison of Sigma anti-5-mC recombinant rabbit monoclonal antibody (clone RM231, Product No. SAB5600040) with 2 competitor antibodies

Comparison of anti-5-mC vs 2 competitor antibodies

ELISA analysis of single stranded DNA. The plate was coated with streptavidin and then biotinylated single stranded unmethylated DNA, 5-Methylcytosine (5-mC) DNA, and 5-Hydroxymethylcytosine (5-hmC) DNA. A serial dilution of anti-5-mC antibodies were used as the primary antibody, and an alkaline phosphatase conjugated anti-rabbit IgG as the secondary antibody.

 

Dot blot analysis of double stranded DNA.

Dot blot analysis of double stranded DNA. The membrane was pre-spotted with 50, 5, and 0.5 ng/dot of double stranded 5-Hydroxymethylcytosine (5-hmC) DNA, 5-Methylcytosine (5-mC) DNA, and unmethylated DNA. The pre-spotted membrane was then blotted with anti-5-mC antibodies.

Comparison of anti-5-hmC Sigma recombinant rabbit monoclonal antibody (clone RM236, Product No. SAB5600041) with 2 competitor antibodies.

ELISA analysis of single stranded DNA.

ELISA analysis of single stranded DNA. The plate was coated with streptavidin and then biotinylated single stranded unmethylated DNA, 5-Methylcytosine (5-mC) DNA, and 5-Hydroxymethylcytosine (5-hmC) DNA. A serial dilution of anti-5-hmC antibodies were used as the primary antibody, and an alkaline phosphatase conjugated anti-rabbit IgG as the secondary antibody.

 

Dot blot analysis of double stranded DNA.

Dot blot analysis of double stranded DNA. The membrane was pre-spotted with 50, 5, and 0.5 ng/dot of double stranded 5-Hydroxymethylcytosine (5-hmC) DNA, 5-Methylcytosine (5-mC) DNA, and unmethylated DNA. The pre-spotted membrane was then blotted with anti-5-hmC antibody.

Immunocytochemical Staining with anti-5-mC (clone RM231)

ICC staining of HeLa cells using anti-5-mC

Immunocytochemical staining of HeLa cells using 0.5mg/mL rabbit monoclonal anti-5-mC (clone RM231, Product No. SAB5600040) antibody (red). Actin filaments have been labeled with fluorescein phalloidin (green). HeLa cells were fixed with Methanol/HAc (3/1) without denaturing DNA, or were fixed with 4% paraformaldehyde and perminized with methanol (−20 °C) before being treated with 2 N HCl for 30 min at 37 °C to denature the DNA.

Immunocytochemical Staining with anti-5-hmC (clone RM236)

ICC staining of HeLa cells using anti-5-hmC

Immunocytochemical staining of HeLa cells using 0.5mg/mL rabbit monoclonal anti-5-hmC (clone RM236, Product No. SAB5600041) antibody (red). Actin filaments have been labeled with fluorescein phalloidin (green). HeLa cells were fixed with 4% paraformaldehyde and perminized with methanol (−20 °C) before being treated with 2 N HCl for 30 min at 37 °C to denature the DNA.

Ordering Information

Product Name Type Clone Product No.
5-methlycystosine Rabbit monoclonal RM231 SAB5600040
5-hydroxymethycytosine Rabbit monoclonal RM236 SAB5600041

 

Sigma-Aldrich also provides other well characterized recombinant rabbit monoclonal antibodies for histone modifications.

Product No. Description
SAB5600009 Anti-Trimethyl-Histone H3 (Lys27) antibody, Clone RM175
SAB5600007 Anti-Acetyl-Histone H3 (Lys4) antibody, Clone RM149
SAB5600008 Anti-Monomethyl-Histone H3 (Lys9) antibody, Clone RM150
SAB5600014 Anti-Dimethyl Histone H3 (Lys79) antibody, Clone RM181
SAB5600015 Anti-Acetyl-Histone H3 (Lys56) antibody, Clone RM179
SAB5600042 Anti-Histone H2A antibody, Clone RM225
SAB5600043 Anti-Histone H2B antibody, Clone RM230
SAB5600044 Anti-Histone H2AX antibody, Clone RM214
SAB5600046 Anti-Histone H4 antibody, Clone RM212
SAB5600048 Anti-Histone H3 K36M Antibody, Clone RM193
SAB5600016 Anti-Phospho-Histone H4 (pSer1) antibody, Clone RM194
SAB5600025 Anti-Phospho-Histone H2A/H4 (pSer1) antibody, Clone RM216
SAB5600026 Anti-Phospho-Histone H3 (Ser10) antibody, Clone RM163
SAB5600037 Anti-Phospho-Histone H2B (Ser14) antibody, Clone RM238
SAB5600027 Anti-Phospho-Histone H3 (Thr3) antibody, Clone RM159