Gel retardation - (Oct/25/2005 )
A about a week ago (17th Oct) we were introduced to the concept of utilising a gel retardation assay in which we (myself and my colleagues) cloned several partial cDNA's coding for a DNA binding protein into an expression vector. This was to test whether the encoded portions of the protein would bind to a specific response element (that the natural protein binds to the genomic DNA). We found that the partial cDNA clones extend for different distances towards the 5' end primer of the gene.
We then transcribed and translated these cDNA clones in vitro. We then mixed the protein translation products with a 32P labelled genomic DNA fragment. The intention of this was to retard the migration (lanes 2, 3 and 4).
When cDNA clones 3 and 4 were mixed together before in vitro transcription and translation, we observed the appearance of 3 retarded bands in the gel retardation assay. Out lecturer gave us some very open ended questions to answer, which he did not detail the length of depth of the answers.
I was with my colleagues, my friend, known as 'superfoot' has posted a thread previously regarding the questions.
In response to the pictures, they are on the attachment.
Here are the questions that were given to us incase you didnt know:
a) Why was the retarded bands at different position in the gel?
I believe the retarded bands were at different positions in the gel because the different bands had different amounts of nuclear protein attached to them, but is there more i can add to it?
Where in the cDNAs in the sequence coding for the DNA- binding domain located?
I think they were located between lanes 2 and 4, because they were clearly visible on the gel shift assay. It is probable in between 3 and 4, because there is a much stronger reading. Can anyone help me on this>
c) Why are there three retarded bands when cDNA clones 3 and 4 are mixed together?
What does that tell you about the way in which the protein normally binds to DNA?
Note that when cDNA bands 3 and 4 are mixed together, three retarded bands are visible. I think this was due to the heaviness of the genes and the protein- protein interactions, but i dont know how to elaborate on this question.
HERE IS A REPLY I GAVE YOUR BUDDY THE OTHER DAY. NOW THAT I HAVE SEEN THE PICTURE, I DO NOT CHANGE ANY OF WHAT I WROTE; THE INTERPRETATION IS THE SAME. DOES THIS MAKE SENSE FOR YOU? BY THE WAY, YOUR INSTRUCTOR IS TRICKY
"are you still looking for the answer?
it would be best if you could attach a picture.
for question c, what do you mean by '3 retarded genes'? do you mean when the two cDNAs, #3 and #4, are mixed together, you see 3 bands in the final blot? if this is the case, think about the possibily of your DNA binding protein forming dimers or trimers with itself. 3 and 4 are basically truncated forms of the same protein, right? I would have to see the band pattern to be sure, but I bet you have binding by a 3-3 dimer, a 3-4 dimer, and a 4-4 dimer, which will not all be quite the same size becuase they have different amounts of one end lopped off.
you can answer question B if you have some specific information about the different cDNA clones. for example, you will see a band with the free oligo, but not with the oligo-protein complex if you have cut off the portion required for binding to the DNA sequence, do you see?
another thing that might make it easy for you, is to note that 'gel retardation' is the ancient name for this technique. the new names are EMSA (electrophoretic mobility shift assay) or gel-shift assay. if you plug these terms into www.google.com you should get some good info. try Pierce's website too; they have good explanations in their literature accompanied by pictures so you can be sure what you are seeing.