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gradstudent 05-25-2009 06:17 PM

New to EMSA
 
Hi,

I am new to EMSAs, basically I want to do an EMSA-western to determine if my protein of interest binds DNA. I plan to IP my protein and a known TF say myc. Since i dont know if my protein binds DNA, I am going double digest genomic DNA with a combination of different restriction enzymes. I will incubate my IPed proteins with the DNA and run it on a native gel and then transfer to nitrocellulose paper and probe with specific ab for western blotting.

I plan to run IPed proteins without DNA as my baseline to detect the protein bands. I hope to see single bands in my IP alone lanes and several bands in my IP+ DNA fragments lane which would represent my protein binding to DNA.

I wanted to know if this protocol was feasible, basically I am looking for yes/no answer to base my future expts (chip-chip etc) on. I would appreciate any input or comments on this!

Thanks

tantecbiosystems 08-02-2010 01:31 AM

Re: New to EMSA
 
One thing to consider, if your protein has a lot of + charges it may stick to DNA nonspecifically. You will need a competitor perhaps polyI:C. Do you see your protein in the nucleus by fluorescence? It would give you some confidence that it is a potential DNA binding protein. What is the size of your DNA, genomic DNA of any size will be too big to get into the gel with your protein. Most EMSA probes are 10 to 20 bp. Try running your sample on an affinity DNA column in conjunction with you antibodies, increased salt may be a simpler way to go

mmorgan 06-24-2011 07:42 PM

Re: New to EMSA
 
I've also been trying to figure out whether a protein of interest has DNA binding activity. Besides cutting up genomic DNA, you can also synthesize a library of sequences using oligonucleotides. This has the advantage that the oligos should be the same length. If you can purify your protein, you can also label the oligos and try to clone the binding sequences.


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