I am doing an unwinding assay for my protein with a fork substrate and my protein-DNA complex is stuck in the wells. My quench to stop the reaction has 0.5% SDS, unlabelled strand, EDTA and glycerol. I tried the following:
1. lower % gels.
2. Proteinase K treatment - RT and 37C incubation
3. Increasing the concentration of SDS in Quench to 5%.
4. Heating the sample to 60C after quenching the reactions.
None of the above worked for this protein. Does anybody have a different suggestion ?
Thanks
Another problem that can have an effect on protein and presumably protein-DNA complex migration through the gel is pH. If the protein or complex has a theoretical PI close to or above the pH then the protein will be uncharged or positively charged and may not migrate beyond the wells. While sds should help this to some degree as the complex enters the gel the sds in the sample may lose effectiveness since the gel and buffer for the native system lack sds. You could try adding a small negatively charged tag to the protein terminus to aid entry and migration through the gel.
@ Reed : Thanks ! The pI of my protein is less than the pH of the running buffer, so it would move to the positive electrode. I don't know what will be the pI of my protein-DNA complex though. Maybe the negatively charged DNA is neutralizing the complex ? Adding a tag to the protein is a bit complicated in my case, so maybe I should try something different. But I used up to 5% SDS, so shouldn't that add positive charge to the protein and be enough to migrate on a gel ?
@ Aral : Thanks ! I thought about that initially but all of my DNA is in complex with the protein, so if I load only the supernatant, there will be nothing on the gel. That is why I did not centrifuge. The lowest I tried was an 8% gel.
@ Reed : I am sorry, SDS would add negative charge.
@ Aral : I am using purified protein and commercial DNA. The DNA is 53 + 23 bases and the protein is a hexamer (310 kDa). That was on my mind too, but I don't have one in the lab. Neither a gradient maker.
Are you sure that the one stuck at the well is protein-DNA complex?
If so, digestion with Proteinase K in the presence of SDS may solve the issue.
In my experience, pre-warming of gel or addition of glycerol (2-5%) in gel was helpful
Because it's an unwinding assay, and not a DNA binding assay, follow your reaction by a SDS (0.2% final) and proteinase K (0.5 mg/ml) treatment ( 37 °C for 5 min).
For good protocols, see Patrick Sung's publication. Example: http://www.jbc.org/content/278/45/44331.full
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