Immunoprecipitation (IP)
(5-Hydroxymethylcytidine antibody tested by Methyl DNA immunoprecipitation. DNA (25 pg) derived from the promoter of the APC gene was spiked into 500 ng of human genomic DNA and subjected to the MeDIP procedure using 2 ?g of 5-Hydroxymethylcytidine antibody (5hmC, maroon bars) or 2 ?g of control rabbit IgG (IgG, blue bars). Real time quantitative PCR was performed on the immunoprecipitated DNA and results plotted as % of input DNA. The spiked APC DNA contained either no methylation (DNA), 5-methylcytosine methylation (5-mC) or 5-hydroxymethylcytosine methylation (5-hmC). The 5-hmC antibody shows a 650-fold enrichment for 5-hmC DNA compared to 5-mC DNA or unmethylated DNA.)
Rabbit anti-Human, Mouse 5-Hydroxymethylcytosine Polyclonal Antibody
5-Hydroxymethylcytosine (5-hmC) antibody (pAb)
Reactivity
Human, Mouse
Applications
Dot Blot, Immunofluorescence, Immunohistochemistry, Immunoprecipitation
Purity
Protein A Chromatography
Synonyms
5-Hydroxymethylcytosine; Polyclonal Antibody; 5-Hydroxymethylcytosine (5-hmC) antibody (pAb); anti-5-Hydroxymethylcytosine antibody
Host
Rabbit
Reactivity
Human, Mouse
Clonality
Polyclonal
Isotype
IgG
Purity/Purification
Protein A Chromatography
Form/Format
PBS pH7.5 containing 30% glycerol and 0.035% sodium azide. Sodium azide is highly toxic.
Concentration
1ug/ul (varies by lot)
Applicable Applications for anti-5-Hydroxymethylcytosine antibody
Dot Blot (DB), Immunofluorescence (IF), Immunohistochemistry (IHC), Methyl DNA Immunoprecipitation (MeDIP)
Application Notes
Applications Validated:
MeDIP: 1-2ug per IP
IHC (FFPE): 1ug/ml dilution
DB: 0.2ug/ml dilution
MeDIP: 1-2ug per IP
IHC (FFPE): 1ug/ml dilution
DB: 0.2ug/ml dilution
Conjugation
Methylated
Immunogen
This 5-Hydroxymethylcytidine antibody was raised against 5-hydroxymethylcytidine conjugated to KLH and recognizes 5-hydroxymethylcytosine.
Preparation and Storage
Some products may be shipped at room temperature. This will not affect their stability or performance. Avoid repeated freeze/thaw cycles by aliquoting items into single-use fractions for storage at -20 degree C for up to 2 years. Keep all reagents on ice when not in storage.1ug/ul
Immunoprecipitation (IP)
(5-Hydroxymethylcytidine antibody tested by Methyl DNA immunoprecipitation. DNA (25 pg) derived from the promoter of the APC gene was spiked into 500 ng of human genomic DNA and subjected to the MeDIP procedure using 2 ?g of 5-Hydroxymethylcytidine antibody (5hmC, maroon bars) or 2 ?g of control rabbit IgG (IgG, blue bars). Real time quantitative PCR was performed on the immunoprecipitated DNA and results plotted as % of input DNA. The spiked APC DNA contained either no methylation (DNA), 5-methylcytosine methylation (5-mC) or 5-hydroxymethylcytosine methylation (5-hmC). The 5-hmC antibody shows a 650-fold enrichment for 5-hmC DNA compared to 5-mC DNA or unmethylated DNA.)
Immunoprecipitation (IP)
(5-Hydroxymethylcytidine antibody tested by Methyl DNA immunoprecipitation. DNA (25 pg) derived from the promoter of the APC gene was spiked into 500 ng of human genomic DNA and subjected to the MeDIP procedure using 2 ?g of 5-Hydroxymethylcytidine antibody (5hmC, maroon bars) or 2 ?g of control rabbit IgG (IgG, blue bars). Real time quantitative PCR was performed on the immunoprecipitated DNA and results plotted as % of input DNA. The spiked APC DNA contained either no methylation (DNA), 5-methylcytosine methylation (5-mC) or 5-hydroxymethylcytosine methylation (5-hmC). The 5-hmC antibody shows a 650-fold enrichment for 5-hmC DNA compared to 5-mC DNA or unmethylated DNA.)
Dot Blot (DB)
(5-Hydroxymethylcytidine antibody tested by dot blot analysis. DNA from the Methylated DNA Standard Kit were spotted (indicated in ng on the left) on to a positively charged nylon membrane and blotted with 5-Hydroxymethylcytidine antibody recognizing 5-hydroxymethylcytosine (1 ug/ml dilution). Lane 1: double-stranded DNA 5-hydroxymethylcytosine. Lane 2: double-stranded DNA containing 5-methylcytosine. Lane 3: double stranded unmethylated DNA. Lane 4: single-stranded DNA containing 5-hydroxymethylcytosine. Lane 5: single-stranded DNA containing 5-methylcytosine. Lane 6: single stranded unmethylated DNA.)
Dot Blot (DB)
(5-Hydroxymethylcytidine antibody tested by dot blot analysis. DNA from the Methylated DNA Standard Kit were spotted (indicated in ng on the left) on to a positively charged nylon membrane and blotted with 5-Hydroxymethylcytidine antibody recognizing 5-hydroxymethylcytosine (1 ug/ml dilution). Lane 1: double-stranded DNA 5-hydroxymethylcytosine. Lane 2: double-stranded DNA containing 5-methylcytosine. Lane 3: double stranded unmethylated DNA. Lane 4: single-stranded DNA containing 5-hydroxymethylcytosine. Lane 5: single-stranded DNA containing 5-methylcytosine. Lane 6: single stranded unmethylated DNA.)
Immunohistochemistry (IHC)
(5-Hydroxymethylcytosine (5-hmc) antibody (pAb) tested by Immunohistochemistry Punctate nuclear staining pattern is detected in Formalin-fixed, paraffin-embedded tissue sections from human substantia nigra (midbrain). Top Panel: 5-mC antibody at 1:1000 dilution. Bottom Panel: No primary antibody (2nd step antibody alone))
Immunohistochemistry (IHC)
(5-Hydroxymethylcytosine (5-hmc) antibody (pAb) tested by Immunohistochemistry Punctate nuclear staining pattern is detected in Formalin-fixed, paraffin-embedded tissue sections from human substantia nigra (midbrain). Top Panel: 5-mC antibody at 1:1000 dilution. Bottom Panel: No primary antibody (2nd step antibody alone))
Related Product Information for anti-5-Hydroxymethylcytosine antibody
Short Description: Our 5-Hydroxymethylcytosine (5-hmC) antibody (pAb) was raised in a Rabbit host. It has been validated for use in Dot blot, Immunofluorescence, Immunohistochemistry and Methyl DNA Immunoprecipitation; it has been shown to react with Human and Mouse samples, but the sequence is not species specific so it should react with a wide range of sample types.
Background: We offers two polyclonal antibodies that recognize 5-hydroxymethylcytosine,a whole serum version and apurified IgG version. Both are validated for use in methyl DNA immunoprecipitation (MeDIP). For customers that require the ability to quantitate the amount of IgG in the MeDIP reaction, the purified IgG version is recommended. The whole serum version is very high titre and should be used carefully (0.1-0.5 ul per IP) as not to generate high non-specific background. The whole serum version has been used successfully in immunofluorescence (IF, http://www.ncbi.nlm.nih.gov/pubmed/20639862 Ito et al, 2010), and the purified IgG version is likely to work in this application as well.
DNA methylation is an epigenetic event in which DNA methyltransferases (DNMTs) catalyze the reaction of a methyl group to the fifth carbon of cytosine in aCpG dinucleotide. This modification helps to control gene expression and is also involved in genomic imprinting, while aberrant DNA methylation is often associated with disease. 5-methylcytosine is a modified base that is found in the DNA of plants and vertebrates. A second type of DNA methylation exists,5-hydroxymethylcytosine (5-hydroxy methylcytosine, 5-hmC). This results from the enzymatic conversion of 5-methylcytosine into5-hydroxymethylcytosine by the TET family of cytosine oxygenases. This antibody was developed specifically to distinguish 5-hydroxymethylcytosine from 5-methylcytosine as conventional methods (enrichment by antibody or methyl DNA binding protein, enzymatic digestion and bisulfite sequencing) cannot do so. It is possible that5-hydroxymethylcytosine (5-hmC) represents a pathway to demethylate DNA, as 5-hydroxymethylcytosine is repaired as mismatched DNA and replaced with unmethylated cytosine.
Background: We offers two polyclonal antibodies that recognize 5-hydroxymethylcytosine,a whole serum version and apurified IgG version. Both are validated for use in methyl DNA immunoprecipitation (MeDIP). For customers that require the ability to quantitate the amount of IgG in the MeDIP reaction, the purified IgG version is recommended. The whole serum version is very high titre and should be used carefully (0.1-0.5 ul per IP) as not to generate high non-specific background. The whole serum version has been used successfully in immunofluorescence (IF, http://www.ncbi.nlm.nih.gov/pubmed/20639862 Ito et al, 2010), and the purified IgG version is likely to work in this application as well.
DNA methylation is an epigenetic event in which DNA methyltransferases (DNMTs) catalyze the reaction of a methyl group to the fifth carbon of cytosine in aCpG dinucleotide. This modification helps to control gene expression and is also involved in genomic imprinting, while aberrant DNA methylation is often associated with disease. 5-methylcytosine is a modified base that is found in the DNA of plants and vertebrates. A second type of DNA methylation exists,5-hydroxymethylcytosine (5-hydroxy methylcytosine, 5-hmC). This results from the enzymatic conversion of 5-methylcytosine into5-hydroxymethylcytosine by the TET family of cytosine oxygenases. This antibody was developed specifically to distinguish 5-hydroxymethylcytosine from 5-methylcytosine as conventional methods (enrichment by antibody or methyl DNA binding protein, enzymatic digestion and bisulfite sequencing) cannot do so. It is possible that5-hydroxymethylcytosine (5-hmC) represents a pathway to demethylate DNA, as 5-hydroxymethylcytosine is repaired as mismatched DNA and replaced with unmethylated cytosine.
Product Categories/Family for anti-5-Hydroxymethylcytosine antibody
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Product Notes
The 5-Hydroxymethylcytosine (Catalog #AAA388362) is an Antibody produced from Rabbit and is intended for research purposes only. The product is available for immediate purchase. The 5-Hydroxymethylcytosine (5-hmC) antibody (pAb) reacts with Human, Mouse and may cross-react with other species as described in the data sheet. AAA Biotech's 5-Hydroxymethylcytosine can be used in a range of immunoassay formats including, but not limited to, Dot Blot (DB), Immunofluorescence (IF), Immunohistochemistry (IHC), Methyl DNA Immunoprecipitation (MeDIP). Applications Validated: MeDIP: 1-2ug per IP IHC (FFPE): 1ug/ml dilution DB: 0.2ug/ml dilution. Researchers should empirically determine the suitability of the 5-Hydroxymethylcytosine for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "5-Hydroxymethylcytosine, Polyclonal Antibody" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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