Please check the attached gel pic.
Protein 1 and DNA has normal interaction.
When I incubated protein 1(300nM in all lanes), protein 2(with increasing concentration 500nm to 8uM) and DNA(same concentration in all lanes) some extra shift was observed.
But when I checked the interaction with only protein 2 and DNA then no shift was observed.
can any one explain this, why is this extra shift coming with increasing concentration of protein 2 ? why dose the extra shift look like a smear ?
Either protein 2 interacts with protein 1 to cause this shift or this is a non-specific effect from the protein 2 sample, such as increased ionic strength that alters the electric field and/or the manner in which protein 1 interacts with the DNA. You need to run a buffer control to sort out whether the effect is truly dependent on protein 2. What is the size of the DNA in this assay? Do you have non-specific competitor?
Either protein 2 interacts with protein 1 to cause this shift or this is a non-specific effect from the protein 2 sample, such as increased ionic strength that alters the electric field and/or the manner in which protein 1 interacts with the DNA. You need to run a buffer control to sort out whether the effect is truly dependent on protein 2. What is the size of the DNA in this assay? Do you have non-specific competitor?
In my opinion it could be possible that both proteins and the DNA form a complex but on the other hand it is possible that both proteins bind separtly to DNA and that they compete. You should test whether protein 2 can bind the DNA on its own.
I would recommend performing several titration experiments to determine binding affinities for DNA + Protein1, DNA + Protein2, Protein1+Protein2 as well as DNA + Protein1 + Protein2. An ideal method to run this series of experiments would be MST (MicroScale Thermophoresis). It measures binding affinites in a fast, easy and precise way, free in solution, and for a broad affinity range.
Chapter Aptamer Binding Studies Using MicroScale Thermophoresis
Hi.. Daniel M Cohen, thank you very much for your reply. Protein 1 and protein 2 interaction(protein-protein), this I have to check.
I have some more queries to your reply. Please respond, it will help me to analysis this data.
1. All these reactions are done in same buffer condition(ionic strength), we have well established interaction with protein 1 and DNA, with protein 1 scan as well. If the ionic strength is the issue using same buffer, then protein 1 and DNA--GSA data has no such effect.
2. Can you tell me, what is meant by buffer control.?
3. In this gel pic the used DNA size is 260bp, although i used 115bp(part of this 269bp DNA) for this I'm getting same data(please check attached file for GSA gel pic).?
Protein 1 and DNA have very specific interaction. and what is meant by non-specific competitor..?, Although we are using BSA for all reaction to make DNA and protein interaction more specific.
Hey,
is protein 1 binding to DNA or to a special sequence of DNA? If the later is the case than a non-specific competitor would be DNA which should not be bound by your protein 1. What about the interaction between protein 2 and your DNA? Did you test this sofare?
Hi ..Ulrike Abendroth.. thanx for your reply.
Protein 2 and DNA has no interaction. which we already checked, no shift with DNA and protein 2 reactions.
protein 1 has interaction to specific sequence of DNA.
If you have any more suggestion please reply.
Hi Avijit Das,
I attached a link to a paper which was done in my department were you will find/ see the use of non-specific (DNA) or speciific competitor (DNA). Important for this is, that the competitor (DNA) is not radioactive labeled http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120214
If protein 1 is the protein interacting with the labeled DNA and you get a second shift by adding protein 2 than it should be clear that protein 1 and 2 and DNA form a complex.
Maybe you can now find out if both proteins are interacting.
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