PNNL-13501 - Pacific Northwest National Laboratory

information on binding. The real-time observation under
controlled solution conditions will provide kinetic
information. The renewable surface approach also
permits a wide latitude in selection of surfaces for
biomolecular binding (surface plasmon resonance
requires a metal surface as part of the transduction
mechanism). Finally, the bead format provides a large
surface area sample that can be characterized by a variety
of techniques. In addition, beads with many chemical
functional groups for attaching proteins or DNA are
already commercially available and can serve as the
foundation for creating biomolecular assemblies.
Results and Accomplishments
Renewable Microcolumn System Design
Two custom renewable microcolumn flow cells were
designed and evaluated: a piston flow cell and a rotating
rod flow cell (Figure 1). Both flow cells include a
microcolumn volume of about 1 µL and a path length of
1 mm. The fluidic system allows automated packing of a
microcolumn from a stirred slurry, followed by perfusion
of the column with various sample and wash solutions (all
automated). Optical fibers leading to the flow cell were
used to monitor the microcolumn absorbance during
column delivery and protein binding to the microcolumn
during sample perfusion. Reproducibility of column
packing and absorbance baseline noise was identical for
both devices. However, the rotating rod design
(Figure 1b) was more reliable and rugged over many
months of use.
(a) (b)
Figure 1. Renewable microcolumn flow cells for automated
protein binding studies. (a) Moving piston flow cell;
(b) rotating rod flow cell. Both designs monitor the UV-VIS
absorbance through microcolumns with a 1-mm path length
and 1-µL microcolumn volume.
42 FY 2000 Laboratory Directed Research and Development Annual Report
Measurement of Protein-Protein Interactions
The detection of the binding of multiple proteins was
demonstrated using the renewable microcolumn system.
Microcolumns that are 1 µL in volume were
automatically formed from a slurry of Sepharose beads.
Sepharose beads were derivatized with Protein G, and
absorbance spectra were collected during in situ binding
of human IgG and goat anti-human-IgG. The human IgG
and the anti-IgG were labeled with different fluorescent
dyes. Chicken IgG which will not bind to the Protein G
beads was used as a control to see if nonspecific binding
occurs (no nonspecific binding was observed). The
absorbance spectra were collected over time and showed
that the ultraviolet absorbance (280 nm) corresponded to
the dye absorbance (496 or 557 nm) expected upon
protein binding (Figure 2). The proteins were removed by
perfusion with 0.1 M HCl. The data showed that the
renewable microcolumn system can detect in situ binding
of multiple proteins. We monitored the binding of three
protein complexes, and expect that the system would be
suitable for investigating complexes containing up to
15 to 20 proteins. The detection limit of the renewable
microcolumn system that included a 1-mm path length
and Sepharose 4B beads (Zymed) was estimated to be
0.005 µg protein bound to the beads (as measured using
commercial IgG antibody proteins). However,
fluorescence detection (rather than absorbance) will be
necessary in some cases for increasing sensitivity to allow
detection of proteins with low absorbance. In the next
year of this project, we will incorporate on-column
fluorescence detection.
Measurement of DNA-Protein Interactions
Initial experiments were also conducted to demonstrate
that the renewable microcolumn system can be used to
measure the binding of nucleotide excision repair proteins
(RPA and XPA) onto DNA fragments. These proteins are
part of a complex set of proteins that are involved in
repairing damaged DNA, an important biological process
in which the details of the formation are not well
understood. Sepharose beads were derivatized with
fluorescently labeled single-stranded DNA fragments, and
absorbance measurements were collected during in situ
binding of RPA protein and XPA protein perfused
sequentially over the microcolumn. The proteins were
removed by washing with 0.1 M HCl, and the labeled
oligonucleotide remained attached to the beads. Initial
results show that both RPA and XPA bind to the singlestranded
DNA. We are continuing these studies to
investigate protein binding onto both single-stranded and
double-stranded DNA that is immobilized onto beads.