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Protein-DNA interaction... - ... how to discover new interactions. (May/26/2010 )

Hallo to everyone...
I give to you e brief description of my problem...
I have the suspect that the protein that I'm working with, MAY have (it is not sure at all!) some interaction with DNA.
I've found protocols who describe how to study "the interactions" (but they already are sure of the fact that the interactions ARE there!!!)

Which is the fastest way to check if a proteins have or not interactions with DNA?

Any suggestion/protocol/experience?

Thank you in advance

-Alinor19-

Alinor19 on May 26 2010, 03:48 PM said:

Hallo to everyone...
I give to you e brief description of my problem...
I have the suspect that the protein that I'm working with, MAY have (it is not sure at all!) some interaction with DNA.
I've found protocols who describe how to study "the interactions" (but they already are sure of the fact that the interactions ARE there!!!)

Which is the fastest way to check if a proteins have or not interactions with DNA?

Any suggestion/protocol/experience?

Thank you in advance


i dont know if this is of any use to you..
We used to do DNA quantitation in a sample which has protein using Picogreen method. One of our protein was a DNA-binding protein so it used to bind DNA (quite obviously) and the florescence signal used to diminish due to this interction. We were not aware of this so what we did was we treated he protein sample with Proteinase K and then did the assay (of course with all the relevant controls). we got enhanced signals and so we were sure that there was some kind of an interaction with the Protein!!!
Best luck!!!

-Prep!-

i dont know if this is of any use to you..
We used to do DNA quantitation in a sample which has protein using Picogreen method. One of our protein was a DNA-binding protein so it used to bind DNA (quite obviously) and the florescence signal used to diminish due to this interction. We were not aware of this so what we did was we treated he protein sample with Proteinase K and then did the assay (of course with all the relevant controls). we got enhanced signals and so we were sure that there was some kind of an interaction with the Protein!!!
Best luck!!!



First of all thank you foir your hint! It may work. Nevertheless I have a question for you (I hope I don't bother you too much!)

1) Which kind of DNA did you have? Plasmid, genomic, digested, fragmented (sonicated or similar).... I ask this because I don't know what should I use to check this potential binding... Obviously I can't use intact genomic DNA (too much viscous), but having no idea of the potential binding site I've got some trouble in choosing a "starting DNA material" (concerning the protein I've got it available pure, so that is not a problem).

Thank you again

-Alinor19-

Alinor19 on May 26 2010, 06:49 PM said:

i dont know if this is of any use to you..
We used to do DNA quantitation in a sample which has protein using Picogreen method. One of our protein was a DNA-binding protein so it used to bind DNA (quite obviously) and the florescence signal used to diminish due to this interction. We were not aware of this so what we did was we treated he protein sample with Proteinase K and then did the assay (of course with all the relevant controls). we got enhanced signals and so we were sure that there was some kind of an interaction with the Protein!!!
Best luck!!!



First of all thank you foir your hint! It may work. Nevertheless I have a question for you (I hope I don't bother you too much!)

1) Which kind of DNA did you have? Plasmid, genomic, digested, fragmented (sonicated or similar).... I ask this because I don't know what should I use to check this potential binding... Obviously I can't use intact genomic DNA (too much viscous), but having no idea of the potential binding site I've got some trouble in choosing a "starting DNA material" (concerning the protein I've got it available pure, so that is not a problem).

Thank you again


Hi alinor
actually it is highly improbable that ours was an intact DNA and i say that because it came after many process chromatographic steps so the DNA being intact is not possible. Also it could have been plasmid as well as genomic.. which i claim cause we did Q PCR for both and got signals!!! Picogreen binds any double stranded and does not distinguish between a genomic or plasmid DNA and it can bind if i m n wrong even a 30-50 base pair frangent of ds DNA!!!
Hope this is of some help!!
Best luck!!

-Prep!-

Alinor19 on May 26 2010, 11:18 AM said:

Hallo to everyone...
I give to you e brief description of my problem...
I have the suspect that the protein that I'm working with, MAY have (it is not sure at all!) some interaction with DNA.
I've found protocols who describe how to study "the interactions" (but they already are sure of the fact that the interactions ARE there!!!)

Which is the fastest way to check if a proteins have or not interactions with DNA?

Any suggestion/protocol/experience?

Thank you in advance


Hi there!

If you know where your protein could be interacting with the DNA (some promoter, some DNA consensus site...) you can perform an EMSA assay.

You can as well produce primers fused to Biotin, and use them to amplify the region you think your protein is binding to. Then you incubate your lysate with the biotinilated-DNA and pull down with Streptavidin-magnetic beads. Then you just perform a WB against your protein of interest to check if your protein is binding the region you amplified with your primers

-laurequillo-

laurequillo on Wed May 26 12:40:27 2010 said:


Hallo to everyone...
I give to you e brief description of my problem...
I have the suspect that the protein that I'm working with, MAY have (it is not sure at all!) some interaction with DNA.
I've found protocols who describe how to study "the interactions" (but they already are sure of the fact that the interactions ARE there!!!)

Which is the fastest way to check if a proteins have or not interactions with DNA?

Any suggestion/protocol/experience?

Thank you in advance


Hi there!

If you know where your protein could be interacting with the DNA (some promoter, some DNA consensus site...) you can perform an EMSA assay.

You can as well produce primers fused to Biotin, and use them to amplify the region you think your protein is binding to. Then you incubate your lysate with the biotinilated-DNA and pull down with Streptavidin-magnetic beads. Then you just perform a WB against your protein of interest to check if your protein is binding the region you amplified with your primers


Hi everyone!
I'm going to get involved in this discussion as I am sitting in the same boat as Alinor19. I have exactly the same problem, ie I have a protein I suspect binds to DNA but no more information than that. So no known binding sites or so. There is only one publication with this gene and not more and that paper is about its expression pattern. The domains or structure of the protein is not known either. Everything about this gene/protein is predicted or unknown. So how do you find whether it is binding DNA or not?

One way I was thinking of was to do a ChIP and at the end when I have reversed the cross-linking and everything, I could just measure the amount of IP:ed DNA. This of course requires a negative control, ie ab for a protein not known to bind DNA, and the whole thing assumes that the used antibody is suitable for ChIP. What do you guys think? Or do you have any better suggestions?

-MehMot-

MehMot on Fri Sep 3 15:56:13 2010 said:


laurequillo on Wed May 26 12:40:27 2010 said:


Hallo to everyone...
I give to you e brief description of my problem...
I have the suspect that the protein that I'm working with, MAY have (it is not sure at all!) some interaction with DNA.
I've found protocols who describe how to study "the interactions" (but they already are sure of the fact that the interactions ARE there!!!)

Which is the fastest way to check if a proteins have or not interactions with DNA?

Any suggestion/protocol/experience?

Thank you in advance


Hi there!

If you know where your protein could be interacting with the DNA (some promoter, some DNA consensus site...) you can perform an EMSA assay.

You can as well produce primers fused to Biotin, and use them to amplify the region you think your protein is binding to. Then you incubate your lysate with the biotinilated-DNA and pull down with Streptavidin-magnetic beads. Then you just perform a WB against your protein of interest to check if your protein is binding the region you amplified with your primers


Hi everyone!
I'm going to get involved in this discussion as I am sitting in the same boat as Alinor19. I have exactly the same problem, ie I have a protein I suspect binds to DNA but no more information than that. So no known binding sites or so. There is only one publication with this gene and not more and that paper is about its expression pattern. The domains or structure of the protein is not known either. Everything about this gene/protein is predicted or unknown. So how do you find whether it is binding DNA or not?

One way I was thinking of was to do a ChIP and at the end when I have reversed the cross-linking and everything, I could just measure the amount of IP:ed DNA. This of course requires a negative control, ie ab for a protein not known to bind DNA, and the whole thing assumes that the used antibody is suitable for ChIP. What do you guys think? Or do you have any better suggestions?


yeah, but one of the problems I see with your approach is that you wont be able to distinguish between direct interactors and proteins that do not bind directly to the DNA but they are in a complex which binds.

-laurequillo-