Tuesday, June 15, 2010

Activation of Transcription Initiation II

Does the Protein Bind DNA?
To determine what DNA sequence a specific protein binds, an electromobility shift assay, or EMSA, is implemented.  This method involves labeling DNA fragments with the sequence of interest and then mixing this DNA sequence with the DNA-binding protein.  We can then run an acrylamide gel with and without the DNA-binding protein, and if the protein does bind the DNA, we should note a shift in the mobility of the DNA, as detected by autoradiography.  Namely, the DNA will become heavier, with the protein bound to it, meaning that it will not travel through the gel as quickly.  Therefore, we should see a shift up.  If the protein does not bind the specific DNA of interest, we should note that the two bands migrate through the gel at the same rate.   We can further test the specificity of the interaction by adding unlabelled competitor DNA:  if we still note a shift in the band, the DNA-binding protein is very specific to the DNA sequence of interest. 

Another method to determine if a protein binds to a DNA sequence is to perform a chromatin immunoprecipitation, which is an extension of an immunoprecipitation.  This technique involves several steps, starting with crosslinking of proteins and DNA.   If the DNA-binding protein is located on the DNA, it will be crosslinked with the DNA (via formaldehyde).  Post-crosslinking, the DNA is sheared using sonication (enzymes can also be used), and the protein of interest is immunoprecipitated.  Crosslinking is then reversed, and we can purify the DNA that was bound to this protein.  Performing PCR on the purified DNA will indicate if a specific DNA region is associated with the DNA-binding protein.

Chromatin immunoprecipitation simplified
1.      Formaldehyde crosslink the DNA and bound proteins
2.      Sonicate to break up DNA
3.      Perform immunoprecipitation with antibodies against the protein of interest
4.      Reverse crosslinking (via high salt, for example)
5.      Purify DNA that co-immunoprecipitated
6.      Perform PCR with gene-specific primers

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