Electrophoresis of ionic complexes

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    • #14323
      dhkwak
      Participant

      I’ve been performing agarose gels to determine the complexation between two ionic species: one is a polymer approximately 10nm in diameter; the other is 19bp siRNA. In brief, I’ve been running my gels at 100V, and I do not want to see these two species separate; instead, I want to see how they complex together.

      My question is, is it possible that a high voltage (e.g., 100V) will separate these two species from each other during the electrophoresis? What are your thoughts? Thanks ๐Ÿ™‚

    • #103023
      JackBean
      Participant

      I think it could separate them. But look to native electrophoresis, where are often the large supramolecular complexes saved. What conditions do they use?

      What about some linking of these two parts?

    • #103028
      dhkwak
      Participant

      Thanks, JackBean.

      Typically, native electrophoresis is done at 100V or higher (buffer is virtually the same, but there are no reducing agents or detergents).

      Just in case anyone is wondering, the cationic and anionic moiety is protonated amine (polymer) and phosphate (siRNA), respectively.

      I can’t imagine voltage may be too influential but I am just wondering if anyone had any experiences with these types of details. A linker would be ideal but may be problematic in synthesis of the polymer and attachment to the siRNA. Thanks

    • #103034
      canalon
      Participant

      What about treating with paraformaldehyde? That should crosslink your 2 moieties, it can be reversed (by heating >65ยบC) and it is quite simple (add to 0.6% v/v final concentration for 20 minutes at RT and wash by precipitating your siRNA). Then run your samples at different voltage and see if that worked or compare boiled and unboiled samples to see if there is a shift.

    • #103052
      dhkwak
      Participant

      Thanks, canalon.

      That sounds a very good idea. Actually, only one of our polymers consists of a protonated amine group, while the others are quarternized (i.e., RN(CH3)+). However, it might be a good idea to try.

      As you may have guessed, this siRNA-polymer complex is for physiological use, so I’m not sure how paraformaldehyde would effect cytotoxicity, efficacy, and how easily the siRNA would dissociate in the body. Do you happen to suggest any papers that may point me in the right direction? ๐Ÿ™‚

    • #103055
      canalon
      Participant

      You might just crosslink for the gels, not for the regular use. You will lose information about the physiological stability, but considering the short length of crosslinking allowed by PFA you will know if your prepared complex was interacting as expected. Maybe use a nuclease assay type of thing to check that the interaction is done at the correct point, not at random when crosslinked.

    • #103056
      canalon
      Participant

      On second thought the PFA crosslinking is stable in physiological conditions (it is a covalent linkm that needs temp >65ยบC to be destroyed) so if the stable interaction do not bother you , and if you can wash your complex to to remove all the free PFA, the crosslinked complex should be OK.

      And I want ma share of the royalties ๐Ÿ˜‰

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