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To study DNA-protein interaction, I want to do a DNA retardation test by mixing the protein with DNA and afterwarts loading it on an agarose gel to see if the DNA migrates slower. I've found some articles where they do this and they state that they mix the DNA with protein in an 1:8 charge ratio, but I can't really figure out how to interpret this

Could anyone tell me how to calculate a DNA:protein charge ratio? If 1:8 is stated, how should I interpret this?

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  • $\begingroup$ Could you please provide some more context, I'm having a hard time understanding what exactly you want to know. $\endgroup$
    – Mad Scientist
    Commented Jun 15, 2012 at 8:19
  • $\begingroup$ I am guessing this refers to the isoelectric charge of protein molecules vs DNA molecules? $\endgroup$
    – 719016
    Commented Jun 15, 2012 at 8:56
  • $\begingroup$ @Mad Scientist To study DNA-protein interaction, I want to do a DNA retardation test by mixing the protein with DNA and afterwarts loading it on an agarose gel to see if the DNA migrates slower. I've found some articles where they do this and they state that they mix the DNA with protein in an 1:8 charge ratio, but I can't really figure out how to interpret this $\endgroup$
    – Karen
    Commented Jun 15, 2012 at 12:43

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This is really sounds like a guideline, a starting place, not necessarily your best protocol.

In practice its hard to be more specific as MS says. The pH of the buffer will affect the charge of the protein, and to a lesser extent the DNA.

Also the protein may have an ideal pH for binding which you could guess is in the range of 7.0-7.4, but might find that the strongest binding depends on unexpected pH and salt concentrations, or the addition of a small molecule.

The affinity of araC for instance is greatly increased by the presence of arabinose in sufficient concentration.

lac Repressor, in contrast releases itself from DNA in the presence of IPTG or lactose.

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