Maybe you could solve the problem using a dye exclusion assay instead of gel retardation assay. The principle is simple: a DNA dye (GelRed, PicoGreen, ethidium bromide) will have a harder time sticking to the DNA depending on how well your nanoparticles packs the DNA. Therefore you will notice a decrease in fluorescence for the nanoparticles+DNA+dye compared to the naked DNA + dye. This could be measured on a plate reader and is a more objective method.

It works quite well for polymeric particles which packs DNA.

Dear Simon,

The gel electrophoresis of the gold nanoparticle with DNA may give you a smear, not a clear band. You can run one well only DNA and one well only nanoparticles. As suggested by Utsa Roy you can confirm the NP and dye binding.

To avoid the smear, you can check the concentration based kinetic of DNA and nanoparticles. High concentration of DNA and low concentration of NP will give you two different bands while a high concentration of NP and low concentration of DNA will not move quickly from the well. In this way, you can easily optimize the exact amount of NP and DNA binding.

For a rough idea, you can check my paper https://www.researchgate.net/publication/319647058_Efficient_intracellular_delivery_of_biomacromolecules_employing_clusters_of_zinc_oxide_nanowires

In supplementary data figure 3, I have performed EMSA, although it is not for NP, can give you an idea of the smear and the clear band.

Hope this information is helpful for you.

Dear Gentlemen,

Thank you for your answers.

Prashant Sharma your paper is very helpful and interesting. I look forward to reading it in its entirety and in more depth. Thanks for your advice.

If you coat the DNA-gold complex with polymer (PEG) you will get clear and distinct bands. Bare gold is mostly citrate capped, so it already has significant amount of charge from citrate group, the charge contribution from DNA will be less significant. But if you coat the gold complex with PEG, 100 % charge contribution will come from DNA only.

I got this for the case when there is only 1 or 2 DNA per gold particle.

+ No DNA particle: remains in the well

+ 1 and 2 DNA particle: have slight shift

+ Citrate cap gold : move fastest b/c most charged

Good luck.

Maybe you could solve the problem using a dye exclusion assay instead of gel retardation assay. The principle is simple: a DNA dye (GelRed, PicoGreen, ethidium bromide) will have a harder time sticking to the DNA depending on how well your nanoparticles packs the DNA. Therefore you will notice a decrease in fluorescence for the nanoparticles+DNA+dye compared to the naked DNA + dye. This could be measured on a plate reader and is a more objective method.

Hi Mr Simon

I have a problem like yours. can you please help me and say that how you run a gel contrasting gold nanoparticles, DNA functionalised gold nanoparticles and the DNA used to functionalise the gold nanoparticles?

thanks for time.