|DNA Dot Blot||1:1000|
IMPORTANT: This protocol employs an atypical fixation and denaturation strategy with which only certain targets are compatible. Where appropriate, this protocol will be linked to its validated antibody under the Product Information banner on the product-specific webpage.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
NOTE: All subsequent incubations should be carried out at room temperature (20-25°C) unless noted otherwise.
Rinse three times in 1X PBS for 5 min each.
NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Section C, Step 8.
Counterstain as appropriate.
NOTE: When including fluorescent cellular dyes in your experiment (DNA dyes, etc.), please refer to the dye product page for its recommended protocol. View our listing of cellular dyes validated for use in immunofluorescence.
posted December 2015
revised December 2020
Protocol Id: 865
Note: This protocol is written for spotting fragmented, purified genomic DNA (titration of 1000 ng, 500 ng, 250 ng, 125 ng, 62.5 ng, 31.25 ng, and 15.625 ng) onto a positively charged nylon membrane using a 96-well dot blotting apparatus. Depending on the source and type of DNA, more or less DNA may be required for detection with the antibody.
• Purify genomic DNA using a genomic DNA purification protocol or kit and sonicate
genomic DNA to generate fragments between 200 and 500 bp. DNA fragment size
can be analyzed by gel electrophoresis on a 1% agarose gel with a 100 bp DNA
• Cut a piece of nylon membrane to the size of the dot blot manifold.
• Wet nylon membrane with 10x SSC Buffer.
• Dry membrane by placing it in 96-well dot blot apparatus and applying vacuum.
NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following incubation and declines over the following 2 hr
posted November 2015
Protocol Id: 804
All Species Expected
Monoclonal antibody is produced by immunizing animals with 5-hydroxymethylcytidine.
Methylation of DNA at cytosine residues is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting, and mammalian development (1,2). 5-methylcytosine is a repressive epigenetic mark established de novo by two enzymes, DNMT3a and DNMT3b, and is maintained by DNMT1 (3, 4). 5-methylcytosine was originally thought to be passively depleted during DNA replication. However, subsequent studies have shown that Ten-Eleven Translocation (TET) proteins TET1, TET2, and TET3 can catalyze the oxidation of methylated cytosine to 5-hydroxymethylcytosine (5-hmC) (5). Additionally, TET proteins can further oxidize 5-hmC to form 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), both of which are excised by thymine-DNA glycosylase (TDG), effectively linking cytosine oxidation to the base excision repair pathway and supporting active cytosine demethylation (6,7).
TET protein-mediated cytosine hydroxymethylation was initially demonstrated in mouse brain and embryonic stem cells (5, 8). Since then this modification has been discovered in many tissues, with the highest levels found in the brain (9). While 5-fC and 5-caC appear to be short-lived intermediate species, there is mounting evidence showing that 5-hmC is a distinct epigenetic mark with various unique functions (10,11). The modified base itself is stable in vivo and interacts with various readers including MeCP2 (11,12). The global level of 5-hmC increases during brain development and 5-hmC is enriched at promoter regions and poised enhancers. Furthermore, there is an inverse correlation between levels of 5-hmC and histone H3K9 and H3K27 trimethylation, suggesting a role for 5-hmC in gene activation (12). Lower amounts of 5-hmC have been reported in various cancers including myeloid leukemia and melanoma (13,14).
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