Biology Reference
In-Depth Information
Biotinylated avi-tag UvrA
streptavidin Qdot conjugate
Quantum dots
(Qdots)
HA UvrB antibody sandwich
Qdot conjugate
F
IG
. 4. Quantum dot conjugation strategies. To ensure that there is no cross talk between
different quantum dots, a differential conjugation strategy is important. Two approaches that were
used for UvrA and UvrB conjugations are highlighted. UvrA (left) was bound to quantum dots using
a short peptide sequence extension, which is biotinylated either endogenously or more efficiently
using biotin ligase. UvrB could be differentially labeled by using an HA tag, which can be labeled
using an antibody sandwich strategy.
the protein does not interfere with its DNA binding or interaction with other
proteins.
16
UvrB was conjugated to Qdots by placing antibody recognition
sequence of hemagglutinin, HA tag, on its N terminus. Specific high-affinity
mouse monoclonal antibodies bind to the HA tag. Qdots coated with goat F
(ab
0
)2 antimouse IgG conjugates are then used to make an ''antibody sand-
wich''.
85
Using this dual labeling approach, both proteins could be observed
simultaneously as UvrA loads UvrB onto damaged sites.
16
As many re-
searchers use polyhistidine tags and nickel chromatography to purify and
tag mammalian proteins, Piehler and coworkers have developed a chemical
tag that binds to six or more histidines.
88
They have used this approach to
effectively label single molecules both
in vitro
and
in vivo.
89-91
Application of these methods allows the physical biochemistry of the DNA
search to be examined. This is a problem that is pertinent not only to the
mechanism of NER, but also to all protein systems that interact with DNA, and
which can be addressed only now with the development of single-molecule
methods. Specifically, using direct imaging (see approach 4 above), the first
glimpses of how the bacterial NER proteins find lesions have been obtained
after over four decades since the discovery of genes.
