I am conducting a 2% agarose gel electrophoresis to analyze polymeric nanoparticle encapsulation of Cas9 RNP. My sample set has a charge range from 0 to +10 mV, and I am observing that the more positively charged samples exhibit significant backward migration off the gel. While my gRNA and Cas9 RNP controls show excellent separation with clear, distinct bands, I encounter issues after adding the polymeric nanoparticles for encapsulation. In these cases, I see no defined bands—only backward streaking. I am seeking advice on how to prevent this backward migration or improve band visibility and resolution. Any suggestions would be greatly appreciated. Thank you!
To begin, it's important to ask yourself a few critical questions:
What is the purpose of this experiment? What are you aiming to analyze and determine? What should the anticipated results look like?
Regarding your electrophoresis...
Can you clarify what you mean by "My sample set has a charge range from 0 to +10 mV"? Are you referring to pristine polymeric nanoparticles or nanoparticles complexed with RNA?
It's logical that pure RNA, being negatively charged, migrates towards the cathode. Similarly, positively charged entities will migrate towards the anode. Therefore, if your pristine nanoparticles carry a positive charge, they will indeed migrate in the opposite direction or "backwards".
When you mix positively charged nanoparticles with negatively charged RNA, the RNA will likely electrostatically attach to the nanoparticle surface. As a result, the charges will either neutralize or, depending on the ratio of nanoparticles to RNA, take on a slightly negative or positive charge. If the mixture of nanoparticles and RNA does not yield specific bands, it could indicate that the RNA has attached to the nanoparticles, resulting in a net neutral charge. Any excess of positively charged nanoparticles would still migrate "backwards".
However, this interpretation is quite speculative.
To better understand your results:
Have you characterized your nanoparticles both in their pristine state and with RNA added? Have you imaged them using TEM? Have you checked the stability of your nanoparticles and nanoparticle-RNA complexes?
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