Mobility shift assay

Malcolm Campbell malcolm at unity.ncsu.edu
Wed Dec 20 09:52:39 EST 1995


In article <v01510109acfc8d0de7d6@[128.118.22.149]>, rxr21 at PSU.EDU
(Ramesh Raina) writes:

> I am having some trobule with mobility shift assays.
> [stuff deleted]
> I would
> expect the DNA-protein complex with 47 KD protein to migrate faster than
> the complex with 70 KD because the DNA fragment used in both the cases is
> same but protein sizes are significantly different.

Ramesh,

As might be expected for electrophoresis under native conditions, our
experience suggests that the isoelectric point (pI) of the DNA-binding protein
is at least as important in determining the migration of shifted target
in EMSAs as the molecular weight of the protein.  I would hypothesize
that your truncated protein has a much higher pI (i.e. is "more basic/less
acidic") than your full length protein.  As such, the truncated protein is more
protonated and therefore has a "more positive" charge than your full
length protein.  As a consequence, your truncated protein migrates at a
lower rate during electrophoresis.  The net result is that the two
proteins appear to be responsible for shifts that migrate at the same
rate through the gel.  This is not an unusual finding for many
transcription factors because the transcriptional activation domain
(TAD) is rich in acidic amino acids relative to the DNA-binding domain
(DBD).  Consequently, when the TAD is removed from the DNA-binding
protein, despite its smaller size, it induces shifts that migrate to the
same extent as the full length protein.  We have observed _exactly_ this
phenomena with two transcription factors that we are characterizing.

I hope that this has been somewhat helpful.  Please feel free to respond to me
directly if you've got any questions.

Best regards and best of luck,

Malcolm

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