Help with interpreting PAGE (polyacrylimide gel electrophoresis) result

[GoGoGadget]

Homework Statement

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!

Homework Equations

N/A.

The Attempt at a Solution

See above.

 

[Ygggdrasil]

I'm not sure I understand your diagram based on the information you gave us. Are the fragments A-C DNA or protein? What are the bands supposed to represent? Is the DNA radiolabeled and you are detecting that or is the gel stained for protein? Why is there a band in lane 1 if it supposedly contains nothing?

 

[GoGoGadget]

I'm not sure I understand your diagram based on the information you gave us. Are the fragments A-C DNA or protein? What are the bands supposed to represent? Is the DNA radiolabeled and you are detecting that or is the gel stained for protein? Why is there a band in lane 1 if it supposedly contains nothing?

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!

 

[Ygggdrasil]

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.

Are you sure the proteins are radiolabeled? Generally in a gel shift assay, only the DNA is labeled and you look at how its mobility changes as you add different proteins to the DNA.

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.

Again, see above. You have to explain why the mobility of the DNA changes when the full length protein is present but not when any of the protein fragments are present.

For the other questions, I'm not sure I understand the diagram for the protein fragments. If I am reading it correctly, fragment 1 contains the N-terminal region of the protein, fragment 2 contains the same region as fragment 1 plus additional sequences, and fragment 3 contains the middle of the protein but lacks both the N-terminal and C-terminal regions. Is this correct?

 

[epenguin]

I'm not sure I understand your diagram based on the information you gave us. Are the fragments A-C DNA or protein? What are the bands supposed to represent? Is the DNA radiolabeled and you are detecting that or is the gel stained for protein? Why is there a band in lane 1 if it supposedly contains nothing?

Yes the original post was lacking some information, suce as that itnisnthenDNA that is labelled and also that mobility is downwards in the fig.

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!

This sound as if it could make sense, but we know nothing about what zones of the protein fragments 1, 2 and 3 are.