DNA dot-blot assay was performed as described previously with some modifications19 (link), 31 , 58 (link). Briefly, genomic DNA was spotted on a nitrocellulose membrane (Whatman). The membrane was placed under an ultraviolet lamp for 20 min to crosslink DNA, and then blocked with 5% milk in TBS-Tween20 for 1 hr, followed by incubation with the anti-5mC or anti-5hmC antibody at 4°C overnight. After incubation with an HRP-conjugated secondary antibody (GeneScript) for 1 hr at RT, the membrane was washed with TBS-Tween 20 for three times and then scanned by a Typhoon scanner (GE Healthcare), and the quantification of 5mC or 5hmC was done by Image-Quanta software (GE Healthcare).
Image quanta software
Manufactured by GE Healthcare
Image-Quanta software is a digital imaging solution developed by GE Healthcare. It provides advanced image processing and analysis capabilities for medical and scientific applications. The software enables users to capture, store, and analyze high-quality digital images from various imaging modalities.
Automatically generated - may contain errors
4 protocols using image quanta software
1
DNA Dot-Blot Assay for 5mC and 5hmC
2
Quantitative Dot-Blot Assay for 5-hmC
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For dot-blot assays, we followed the procedures described previously.37 (link) Briefly, genomic DNA was spotted on nitrocellulose membranes. The membrane was baked at 80 °C and then blocked with 5% skimmed milk in TBST for 1 h, followed by the incubation with the anti-5-hmC antibody overnight at 4 °C and HRP-conjugated anti-rabbit IgG secondary antibody for 1 h at room temperature. After washing three times with TBST, the membrane was treated with ECL and scanned by a Typhoon scanner. The quantification of dot-blot was done by Image-Quanta software (GE Healthcare).
3
Quantifying Genomic 5-Hydroxymethylcytosine
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Genomic DNA was extracted using phenol chloroform method and quantified using NanoDrop. DNA was serial diluted, spotted on a nitrocellulose membrane and crosslinked with the membrane under ultraviolet lamp. The membrane was blocked in 5% milk and then for subsequent primary and secondary antibodies incubation. The membrane was scanned with a Typhoon scanner (GE Healthcare). The 5 hmC intensity was quantified by Image Quanta software (GE Healthcare).
4
Quantification of 5-Hydroxymethylcytosine by DNA Dot-Blot
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DNA dot-blot assay was performed as described previously with some modifications.25 (link) Briefly, genomic DNA was spotted on a nitrocellulose membrane (Whatman plc, Maidstone, UK). The membrane was placed under an ultraviolet lamp for 20 minutes to crosslink the DNA and then blocked with 5% milk in Tris-buffered saline–Tween 20 for 1 hour, followed by incubation with the anti-5hmC (Active Motif, Carlsbad, CA, USA) antibody at 4°C overnight. After incubation with a horseradish peroxidase-conjugated secondary antibody (GeneScript Biotech Corporation, Piscataway Township, NJ, USA) for 1 hour at room temperature, the membrane was washed with Tris-buffered saline–Tween 20 three times and then scanned by a Typhoon scanner (GE Healthcare, Little Chalfont, UK). To control spotting, blots were stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2). Quantification of 5hmC was done by Image-Quanta software (GE Healthcare).
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