Dr E. Buxbaum
EB15 at le.ac.uk
Thu Jul 17 05:22:09 EST 1997
sarafian at njmsa.UMDNJ.EDU (Stefan G. Sarafianos) wrote:
>Anybody has any references or any info about techniques for detecting
>protein-protein interactions with colorimetric or fluoresence techniqes?
>I f the method could be applied for interactions of the protein
>with itself -polymerization- that would be great.
I don't know of any colorimetric techniques. However, fluorescence is an
option. Basically, you label your proteins with 2 different fluorescent
probes, chosen so that the absorbance maximum of label 2 coincides with
the emission maximum of label 1. Under these conditions,
Foerster resonance energy transfer will occur between the probes, if they
are close enough. In other words, exitation of label 1 will result in
emission of label 2. This process depends very much on the distance
between the labels (it's 4th power, if I remember correctly). Any book on
fluorescent methods in biochemistry should give you further detail.
For self association you mix 2 batches of your protein, labelled with the
different probes. Beautiful examples of such studies are the works of
Kosk-Kosicka and of Vanderkooi et al. on Ca-ATPase (JBC 264 (1989)
19495-9, Biochemistry 16 (1976) 1262-7)
Another method used for such studies relies on the formation of "Eximers"
(exited dimers) of suitable labels, see for example Luedi & Hasselbach
(Eur. J. Biochem. 130 (1983) 5-8)
If you have access to an analytical ultracentrifuge, this would provide
the most rigorous approach for such studies, at least for soluble, rather
than membrane bound proteins.
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