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HRP on the Blot - Activity and sensitivity of HRP-mimicking DNAzyme on the blot
The activity of the HRP-mimicking DNAzyme is recovered after the transfer to a nylon membrane. In Fig. 5 different amounts of HRP-mimicking DNAzyme are blotted onto a nylon membrane. After the immobilization of the DNA with NaOH the hemin that is necessary for the catalytic activity of the DNAzyme was incorporated into the G-quadruplex by incubating the membrane in HRP-mimicking DNAzyme buffer supplemented with 100 µM. After this step the catalytic activity was recovered. When still in the denaturing polyacryl amide gel the DNA however is not able to produce reactive oxygen species and thus activate a HRP substrate as for example luminol.
EtBr stained gel could detect amounts to 60 pmol of HRP DNAzyme while the luminol on the membrane could get 10-fold lower.
On top of that Fig. 5 shows that on the EtBr stained gel we can detect amounts to 60 pmol of HRP DNAzyme while we get 10-fold lower with the luminol readout to 6 pmol on the blot.
In Fig. 6 you can see that ssDNA with HRP-mimicking DNAzyme was successfully separated from RNA and DNA of a different size on a denaturing PAGE. In Fig. 6 A the same samples as in B and C were stained with SYBR Gold. Fig. 6 B depicts the gel stained with EtBr that was plotted onto the membrane shown in Fig. 6 C. On the blot the HRP-mimicking DNAzyme that could not be detected on the EtBr stained gel were detected.
T4 Ligase is capable of ligating single strand nucleic acids using a DNA splint (red).