Help with interpreting PAGE data (polyacrylmide Gel Electrophoresis)

[Violagirl]

Hi everyone,

I was going to see if anyone that was familiar with how PAGE is done, if someone would be willing to check to see if my interpretation of the hypothetical data attached is correct? It's a hypothetical experiment where you're testing the DNA-binding domain of a protain to a transcription factor using a native polyacrylamide gel and you have to interpret the results. The hypothetical results are attached below to this post. It's for a homework assignment for a Biology course I'm currently taking and wanted to see if my understanding was correct. Thanks so much!

I know that for native PAGEs that are done, both size and charge of the protein and DNA have to be taken into account for how fast they will move through the gel.

For the first question, it asks about the band in lane 5 and why it appears higher in the gel.

-For it, I speculated that it appeared higher because since no DNA fragment was added to it, it's motion would appear be slower through the gel, hence the higher band. Also that, it perhaps dissociated in the gel to show two size lengths of the protein for the two different bands? I'm not for sure on this though.

For the second question, it asks based on the data, where the DNA-binding domain existed in the transcription factor. And what other domain, besides a domain for DNA-binding, must the transcription factor contain? From here, then how it would impact the binding of the transcription factor to the DNA and where this same domain is located within the protein?

-I thought that because a series of bands are shown in Lanes 6 and 8, that the binding domain must be located on fragments 1 and 3 of the DNA. And that besides a binding domain, it would also need a Transcriptional Activating domain. A transciptional activating domain impacts where DNA binding will take place based on specific sequences of DNA that control the rate of transcription and the activating domain will promote the actual process of transcription for it to take place. On the protein, the activating domain is located on the N-terminus region of the protein.

For the last question, it asks to explain the appearence of the bands that appear in the middle of the lanes 6 an 8 and why they appear at different positions?

-I wrote that it may have been because the DNA fragments dissociated from the protein while running through the gel. And they are at different positions to account for the different sizes of the DNA fragments as it looked like that Fragment 3 was much larger than 1 and would run slower in the gel.

Any input on this would be so great. Thanks so much!

[CharonY]

There may be something lost in translation, but "testing the DNA binding domain of a protein to a transcription factor" does not really make sense as a transcription factor is generally a protein with DNA binding properties. Thus it would imply a protein-protein interaction. It would help if you clarified what is supposed to bind to what.

I will for now assume that you want to test the binding of a protein (or its DNA binding domain) to a cognate DNA region (essentially an EMSA or electrophoretic mobility shift assay).

Assuming that to be true I have some issues understanding the image. What is in lane 1? It is labelled as no protein, but what is there? Are the fragments in 2-4 referring to specific fragments of the DNA-binding protein? Or does it refer to a DNA-fragment?

On the same note, what precisely is in lane 6-8? Are the fragments referring to DNA now, or to protein fragments? If the fragment are all referring to proteins, where is DNA added?

Also what kind of visualization is shown in the gel? I.e. are only the proteins dyed or are DNA and protein visualized?

I could start guessing and assuming that the fragments are really referring to DNA stretches, but I would prefer if you could clarify. Note that domain refer to specific regions on a protein and not on DNA..

[Violagirl]

I got feedback from my professor on a few points you brought up. The first lane is supposed to contain radioactive labled DNA without the protein. Lanes 2-4 contain radioactive labeled fragments (fragments 1-3) of the protein. Lane 5 contains the full length protein with the radioactively labeled DNA. Lanes 6-8 contain fragments 1, 2, and 3 of the protein with radioactive labeled DNA. The gel used in the hypothetical experiment slows the proteins down based on their size, it's a gel shift assay. The bands are associated with the DNA and protein fragments. I have to answer questions based on the results on what it means.

For question one, for explaining why the band appeared higher in the gel in lane 5, I presumed it was because the full length size of the protein impacted it's motion through the gel as its larger size will slow it's movement in the gel.

For question two, reiterating from what I said earlier, I presume the DNA-binding domain is located on the N-terminus region of the protein since the middle bands appear on lanes 6 and 8 but not in 7. Lane 7, based on where the protein fragment is cut, I thought correlated to the C-terminus region and since no middle band appears (I assume to be the DNA-protein binding complex), that no binding takes place in the C-terminus region. For the other kind of domain that the transcription factor to the DNA must have, along with a DNA-binding region, that it also would need a trans-activating domain. The last part to the question asks where the domain is located in the protein and I thought it was located in the C-terminus region.

For question three, on explaining the presence of the middle bands in lanes 6 and 8, I thought they correlated to the DNA-protein interaction. For why they ran at different points on the gel, I thought it correlated to the different sizes of the protein fragments (slower movement of fragment 3 in lane 8 as it is larger than fragment 1 in lane 6).

I've attached a new diagram to this post. Hope this makes more sense, any feedback on my interpretation of this is appreciated!

Unless I'm wrong and it does indeed bind to both regions (the N-terminus and C-terminus). And that the bands appear in the middle lanes in 6 and 8 because of a dissociation between the fragment and DNA. Which might mean that the transactivating domain appears in the N-terminus region? Any input is very much appreciated, thanks again.

[CharonY]

This makes more sense than the earlier depiction. A thing you have to be sure of is whether only the DNA is labeled, or (as you claim) also the protein fragments.

Based on this the first question you would have to answer is actually what is the lowest band that you see in each lane (is it always the same molecule?). Note that in the text you say that 2-4 contains only labeled protein fragments, whereas the annotations states that there is also DNA there. The difference is quite important. Based on that, why do you see only one band in these lanes?

 

Finally one thing you should be clear about is lane 6-8. In the text you say that it contains one protein fragment plus DNA. However the annotations states that in each also the full-length protein is present. Does it make sense to do it that way?

 

Note another thing, under non-denaturing conditions and in absence of external charges the protein migration does not correlate well with size, even within a gel (you have to things to consider, electrophoretic mobility and retardation by the gel). There is a reason to add SDS in standard protein PAGE. Whose migration are you really monitoring in EMSA?

 

Without these points being clear you will be prone to doing to do guesswork.

Edited February 8, 2013 by CharonY