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EMSA, Electro Mobility Shift Assay

10 Nov

Often simply called a gel shift assay, these are performed to identify the binding of proteins (or other molecules, conceivably) to DNA. The basic idea is that you have a section of DNA in excess supply that contains a sequence that you suspect may be bound by known or unknown proteins. You first label your DNA by incorporation of labelled bases via PCR or end-labeling. This used to be done using radioactive bases, but there are other alternatives available now. For our purposes, I’m going to assume a radioactive label as it is simple to imagine.

Once you have your radiolabelled DNA, you now need to add some proteins that you suspect might bind. This could be isolated protein from a recombinant source or it could be a cell lysate from cells that have been treated in some way. Again, I’ll imagine that we have stimulated cells with something for a progressively longer time (say, 0 minutes stimulation – 30 minutes of stimulation at 5 minute intervals). Finally, after the get runs, you can blot it onto a membrane and expose it to film where the radioactive DNA will light up. Assuming this, I guess we also have a radioactive DNA ladder too

Here’s an example… (remember, this is entirely fictitious)

We stimulate cultured B cells given the survival cytokine, BLyS. At the timepoints indicated above (0-30 minutes), we harvest cells and lyse them in the cold to obtain nuclear fractions. Our DNA is derived from the promoter region of a protein we are interested in and we are wondering if we will see transcription initiation factors assemble.

What we are seeing is a 100bp DNA ladder run with our samples. Keep in mind that the ONLY thing we can see here is labeled DNA. In the first several lanes, none of our DNA is being bound and it is running according to its size (~50bp). At 15 minutes, we start to see the shadow of a band that has shifted our DNA up to an apparent ~700bp. IMPORTANT: DNA ladders are not protein ladders! Also, this is a native protein with unknown charge that is bound to our DNA. All we know is that we are getting binding, we can’t assume knowledge of the exact size of the protein (however, in general, we do see shifts going up as more proteins accumulate)

By 30 minutes, we see an appreciable amount of protein binding our DNA. But how can we know what protein(s) are binding?

One way, if we know what we are expecting, is to use an antibody against that suspected protein. Assuming we have extra lysate + DNA mixture to run a second gel, we could spike our antibody into one tube with DNA and lysate and not into the other (or spike an irrelevant antibody).

If we see that the antibody results in a ‘supershift’ where the DNA/protein band has moved up to a larger apparent size, this means that our antibody is binding its target and that target is binding the labeled DNA.

Let’s assume we suspect that Nf-kB is what is binding our DNA, so we use and Nf-kB antibody to do a supershift assay.

These data support our model and we can now go on to ask new questions.

 
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Posted by on November 10, 2020 in Uncategorized

 

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