Spectrophotometric quantitation of DNA on blots after ethanol-solubilization of the MTT-formazan from anti-digoxigenin-based detection of nucleic acids.
The tetrazolium salt, 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) has recently been established as a substitute for Nitro-blue tetrazolium (NBT) in stain mixtures using antibody-conjugated alkaline phosphatase for the location of proteins on Western blots (Heegaard, 1990). Experiments reported here show that MTT is as sensitive as NBT in digoxigenin-labeled probe localization (on nucleic acid blots) utilizing alkaline-phosphatase-labelled, anti-digoxigenin antibodies. Moreover, as the formazan from MTT is soluble in ethanol, it is shown that spectrophotometric quantitation can be used to estimate the amount of target DNA on dot and Southern blots. For dot blotting, pBR328 was used as the probe and pBR322 as target. For Southern blots, human rDNA was used as the probe and total genomic calf DNA as the target. Staining response was linear over at least six twofold DNA dilutions in both types of blot.
Colgan
D J
DJ
Ken and Yasuko Myer Molecular Evolutionary Biology Unit, Australian Museum, Sydney.
D000906:Antibodies; D015139:Blotting, Southern; D004247:DNA / Q000032:analysis*; D004076:Digoxigenin / Q000032:analysis* / Q000276:immunology; D000431:Ethanol; D005562:Formazans / Q000737:chemistry*; D012680:Sensitivity and Specificity; D012995:Solubility; D013053:Spectrophotometry / Q000379:methods*; D013194:Staining and Labeling; D013778:Tetrazolium Salts / Q000737:chemistry*
D000906:Antibodies; D005562:Formazans; D013778:Tetrazolium Salts; C079782:MTT formazan; D000431:Ethanol; D004247:DNA; D004076:Digoxigenin