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Introduced to Gel Retardation Assay - Today in practical class, our lecturer instructed us in GRA (Oct/20/2005 )

A few days 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.

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

cool.gif Where in the cDNAs in the sequence coding for the DNA- binding domain located?

c) Why are there three retarded genes 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?

These last two questions we cannot seem to answer despite help from our lecturers. We would be most grateful if you could shed some light on my colleagues and I.





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 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.

good luck