Hi, i want to see the binding of a protein to different conformation of ss DNA. Is it possible to do EMSA without purification of my protein. I have my interested protein in both mammalian and bacterial expression plasmid.
If you don't purify the protein, you will need to demonstrate that any band shift is due to that protein. You could do this by comparing extracts from uninduced and induced cells. If the protein is not abundant in the extract of induced cells, however, there may not be enough of it to be able to see the band shift. Also, a crude cell extract could have DNase activity that will degrade your ssDNA. To prevent this, you will have to add enough EDTA to chelate divalent metal ions required by DNases for activity.
If you don't purify the protein, you will need to demonstrate that any band shift is due to that protein. You could do this by comparing extracts from uninduced and induced cells. If the protein is not abundant in the extract of induced cells, however, there may not be enough of it to be able to see the band shift. Also, a crude cell extract could have DNase activity that will degrade your ssDNA. To prevent this, you will have to add enough EDTA to chelate divalent metal ions required by DNases for activity.
thanks for the comment.Can i use ecoli cell extract having expressed mammalian protein or i have to use only mammalian cell extract . Also, for demonstrating binding of protein to ss dna conformation only not to sequence, what conditions i have to use.do i have to maintain buffer condition all the time so that ss dna remain in the appropriate conformation all the time.
Without purifying your protein, you cannot be sure of the results. Which protein caused the shift, for example? I also suspect that you'll lose your analytical signal in the general "noise" of bands on your gel.
You should be able to use an E. coli cell extract as long as your protein is expressed in a soluble form.
To demonstrate that the conformation is relevant for binding, not the sequence, you could try creating a completely different sequence that adopts the same conformation.
It would be advisable to use buffer conditions that favor the appropriate DNA conformation at all times because EMSA is an equilibrium binding experiment.
thanks andrew and adam for comments.Can some one refer me a paper or nice protocol in which they used emsa with ecoli extract to demonstrate mammalian protein binding
You can do EMSA without protein purification. If your mammalian gene was cloned into an E. coli expression vector, then try to verify the expression of the protein on an SDS-PAGE protein gel using a crude extract of the E. coli strain with and without the gene of interest. Also use the strain/empty vector crude extract as a control in your EMSA and if the empty extract does not show any binding then you can be pretty sure that the retardation was caused by your protein of interest. Of course for this, as Adam wrote, you have to have the protein in soluble form in E. coli, but if your expression system is not very strong (i.e. does not produce tons of proteins) there is a good chance having the protein in soluble form. Although not for mammalian protein, my attached paper demonstrate EMSA using E. coli crude extract.
Article Nopaline causes a conformational change in the NocR regulato...
Thanks for the paper. Does any one has more info regarding the recent paper or other technique to show binding of expressed protein to dna
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