5-hmC | 5-hydroxymethylcytosine (polyclonal)
AS16 3166 | Clonality: Polyclonal | Host: Rabbit | Reactivity:Human, Mouse, other species
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Product Information
Immunogen
BSA-conjugated molecule: 5-hydroxymethylcytosine (5-hmC)
Host
Rabbit
Clonality
Polyclonal
Purity
Protein G purified.
Format
Liquid
Quantity
100 ĩl
Storage
Store lyophilized/reconstituted at -20°C; for long term storage Store at -80°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
Tested applications
Dot blot (Dot), ELISA (ELISA), hMeDIP (hydroxymethylated DNA Immunoprecipitation (IP)
Recommended dilution
1 : 200 (Dot), 1 : 500 (ELISA), 2,5 ĩl/IP (hMeDIP)
Reactivity
Confirmed reactivity
Human
Predicted reactivity
Mouse, broad species range
Not reactive in
No confirmed exceptions from predicted reactivity are currently known
Application examples
Application examples
application example
An hydroxymethylated DNA IP (hMeDIP) was performed using the rabbit polyclonal antibody directed against 5-hydroxymethylcytosine. The IgG isotype antibodies from rabbit were used as negative control. The DNA was prepared with the GenDNA module of the hMeDIP kit and sonicated with our Bioruptor® to have DNA fragments of 300-500 bp. 1 μg of human Hela cells DNA were spiked with non-methylated, methylated, and hydroxymethylated fragments. The IP’d material has been analysed by qPCR using the primer pair specific for the 3 different control sequences. The obtained results show that the rabbit polyclonal for 5-hmC is highly specific for this base modification (no IP with non-methylated or methylated C bases containing fragments).
An hydroxymethylated DNA IP (hMeDIP) was performed using the rabbit polyclonal antibody directed against 5-hydroxymethylcytosine. The IgG isotype antibodies from rabbit were used as negative control. The DNA was prepared with the GenDNA module of the hMeDIP kit and sonicated with our Bioruptor® to have DNA fragments of 300-500 bp. 1 μg of human Hela cells DNA were spiked with non-methylated, methylated, and hydroxymethylated fragments. The IP’d material has been analysed by qPCR using the primer pair specific for the 3 different control sequences. The obtained results show that the rabbit polyclonal for 5-hmC is highly specific for this base modification (no IP with non-methylated or methylated C bases containing fragments).
ELISA: to determine the titer, an ELISA was performed using a serial dilution of the rabbit polyclonal antibody directed against 5-hmC in antigen coated wells. The antigen used was BSA coupled to the 5-hmC base. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1: 3 500.
Additional information
Additional information
This serum contains 0,05 % sodium azide
Background
Background
5-hmC | 5-hydroxymethylcytosine is a recently discovered DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. It may have an important roles distinct from that of 5-methylcytosine (5-mC). 5-hmC bases have been identified in Purkinje neurons, in granule cells and embryonic stem cells where they are present at high levels (up to 0,6% of total nucleotides in Purkinje cells.
Product citations
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