It is an example of something we think we understand, often a common phenomenon that we take for granted, but is not as it appears to be. There are many such: why does water (e.g. in the ocean, or a lake) appear to be blue?; why do gel shift assays work?
For the latter (gel shift assays), I remain unconvinced by the explanation in the Wikipedia link - the linear DNA, which is huge, can move through the cross-linked gel, so a globular protein molecule (much smaller) should be able to diffuse away.
Any ideas?
2 comments:
Fried and Crothers, Nucleic Acids Research 1981
This is the seminal gel shift paper that provides some evidence for the cage hypothesis. They conclude that within the gel, the rate at which the protein dissociates from the DNA is slow, as evidenced by slow transfer of the protein to a different strand of DNA also in the gel.
Yes, good suggestion.
When I Googled that paper, this one came up. It is even more on point. However, when you read this paper remember that gels do not really have 'pores' - these are a mathematical fiction to describe the interference with diffusion of (macro)molecules in the highly mobile gel network, not a physical reality. It does not change the validity of their model.
"Molecular sequestration stabilizes CAP- DNA complexes during polyacrylamide gel electrophoresis"
Michael G.Fried* and Gang Liu, Nucleic Acids Research, 1994, Vol. 22, No. 23, 5054-5059.
ABSTRACT
The gel electrophoresis mobility shift assay Is widely
used for qualitative and quantitative characterization
of protein complexes with nucleic acids. Often it is
found that complexes that are short-lived in free
solution (t1, of the order of minutes) persist for hours
under the conditions of gel electrophoresis. We have
investigated the influence of polyacrylamide gels on the
pseudo first-order dissociation kinetics of complexes
containing the E.coli cyclic AMP receptor protein (CAP)
and lactose promoter DNA. Within the gel matrix, kdis
decreased with increasing [polyacrylamide] and the
order of the reaction was changed. In free solution,
kd" was proportional to [DNA]2, while in 5% gels, kdi
was proportional to [DNA]0.3. In gels of [polyacrylamide]
> 10%, kdIu was nearly independent of [DNA]
until fragment concentrations exceeded 0.1 ,uM. Even
in the absence of competing DNA, kdiss(gel) <
kdl,s(solution). These results suggest that the lifetime
of CAP - DNA complexes in free solution is limited by
their encounter frequency with molecules of DNA or
with protein - DNA complexes; some or all of the
stabilization observed in gels may be due to a reduction
in this frequency.
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