I think it is more likely that the failure of the complex to enter the gel is that it is too large. Try a lower percentage gel.
I tried 4% PAGE, but it still remain in the well. Also I tried 0.8% ,1.2%agrose gel, the results is only a minor shift between free DNA and protein-DNA comlex. At last, I tried a PAGE/agrose mixture gel, in this case a more significant shift could be seen. What confuse me is the protein-DNA comlex shift is like a smear. I don't know the reason.
I think the smearing may happen because the protein and DNA are in equilibrium and dissociation of the complex occurs during electrophoresis.
Dan Jin, can you post the protocol for PAGE/agrose mixture gel cause I experience the same problem
Hi，Liudmyla lakovenko, it's simple to prepare the mixture gel
Prepare the 0.8%agrose first, then prepare the PAGE mixture while cooling the agrose. ( do not add APS and TEMED now)
When the 0.8%agrose is cooled, add APS and TEMED to the PAGE mixture , and mix the two solution by 1:1 or other ratio.
Hi,
You use lower pH running buffer than pI, So it makes your protein's net surface positive. Therefore, Is it possible that your proteins cannot run since it is charged as a positive. Normally, running is based on charges between 2 sides, and in denaturing gel, using SDS charges your proteins negatively then they can run from katod to anod in electrophoresis. But in native gel you don't use SDS or other things. Hence, naturally being charged positive could cause this problem? Besides, what is the pH of your buffer which you keep your proteins?
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