Getting good band in the only dna lane but jot getting band in dna-protein sample lanes. I increased the concentration of labelled DNA to 26ng in each sample.
Hello,
Do you mean that you get a shift, but with a smear in the DNA-protein samples ? If so, it may be that the DNA-protein complex is not fully stable and dissociates during migration.
EMSA GEL
1st lane contains only DNA and the rest has both protein and DNA, protein with increasing concentration.
it would be best to see the whole gel, with the complete set of signal of the probe at the bottom. I only see one lane with unbound probe. It seems promising
Are you expecting different complex stoichiometry with increasing the amount of protein? It would seem that the more protein you add, the complex migrates less. To get sharper bands for your protein:DNA complex I would suggest decreasing the amount of salt you have in your incubation mixture (be careful also with how much carry-over you have with increasing the amount of protein). If that doesn't help try a different pH of your incubation and running buffers.
Hi
I had used 0.5x TBE buffer as the running buffer.
For incubation, I used storage buffer and 4x binding buffer.
Also agree with Pierre Béguin
I don't know if this will be your case but If your protein that binds the DNA uses some divalent cation as a cofactor, you can try to add the same concentration of the metal in the gel and running buffer to avoid the dissociation during the running....
you must first show the only probe lane and the binding in the rest of the lanes. If your first lane is the only DNA probe, then the signal itself is very weak, and the bands that you are showing in the rest of the lanes are basically the leftover DNA probe after binding with the protein is completed. I do not think that these bands show interactome complexes. You must run it in a big gel and for long hours (the tracking dye must run at least 4/5th the length of the gel) in cold condition, and at proper voltage to prevent overheating of the gel.
I agree with Cristina, Check if you need any metal ions! I had Mg in my binding buffer. Also can you post the recipie of your Binding buffer and time you incubate before running your gel?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA