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Help with interpreting PAGE data (polyacrylmide Gel Electrophoresis)

DNA-protein binding

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#1 Violagirl



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Posted 03 February 2013 - 10:43 AM

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!

Attached Thumbnails

  • DNA-binding of protein.jpg

Edited by Violagirl, 03 February 2013 - 10:44 AM.

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