Focusing on region D of the Hpy promoter, you generate a radioactively labeled D
ID: 82677 • Letter: F
Question
Focusing on region D of the Hpy promoter, you generate a radioactively labeled DNA fragment containing this sequence. You fractionate extracts derived from the neuronal cell line, mix the fractions with the region D fragment and run these samples on a native gel Which fraction (s) may contain a factor that specifically binds the D region sequence? Which fraction (s) may contain a factor that binds any DNA sequence? Propose an experiment using this same native gel system to differentiate between the possibilities raised in Be sure to include a description of potential results.Explanation / Answer
3C.
The given gel is an EMSA gel (Electrophoretic Mobility Shift Assay).
It is a native PAGE gel.
In this assay, suspected DNA sequences are labelled with radioactivity and incubated with a protein of interest.
The DNA-Protein samples are run on a native PAGE gel. Since the protein is of high molecular weight, it runs slowly in the gel compared to DNA.
If there is an interaction between DNA and protein, the DNA movement will be retarded and appears as a high MW band.
If there is no interaction between DNA and protein, it runs freely withoit being retarded.
In the given gel, two bands (In lanes 3 and 5) are retarded. So, those two DNA sequences are interacting with DNA. But, we can not say which is a specific or nonspecific interaction, without the sizes of protein, DNA and a marker.
(i.e. without positive and negative controls)
3D.
EMSA is only a preliminary invitro result. It does not necessarily imply that the given protein regulates a specific gene expression.
We can use controls to distinguish between specific and non specific interaction.
For example, take a random protein and incubate with the DNA sequences. If the DNA sequence shows retardation in the gel with any other random protein, then it is nonspecific binding.
Most of the times, nonspecific binding is weaker compared to specific binding. So, we can use previously known DNA sequence that shows specific binding to compete out nonspecific binding. This is called Cold-hot competition assay.