PNNL-13501 - Pacific Northwest National Laboratory

Anti-Human lgG
Human lgG
Protein G
Sepharose Bead Surface
(a)
The renewable microcolumn format allows rapid,
automated testing of many experimental parameters such
as solution concentrations, bead surface chemistries, and
the order of perfusion events.
Binding Kinetics
We evaluated kinetic models for modeling protein
binding within the renewable microcolumn system in
order to use the system to obtain quantitative kinetic
information. The CLAMP computer program (University
of Utah) for bimolecular interaction analysis was used to
estimate the kinetic constants that can be investigated
using the renewable microcolumn system. This analysis
indicated that the renewable microcolumn system could
be used to investigate binding reactions with equilibrium
constants ranging from 10 6 to 10 12 , which covers many
biomolecular interactions of interest.
Summary and Conclusions
0628 m
relative absorbance
0.18
0.13
0.08
0.03
Two automated renewable microcolumn systems with
on-column absorbance detection were successfully
designed, assembled, and tested. The automated
systems were used to detect multiple protein binding
events and also DNA-protein interactions. The
advantages of this approach for studying biomolecular
interactions are
280 nm
496 nm
557 nm
Chicken IgG
Human IgG-
FITC
-0.02
0 300 600 900
time (s)
1200 1500 1800
1. automated renewal of the sensing surface eliminates
problems with surface fouling
2. the renewable microcolumn format and automated
fluidics allow rapid, automated testing of many
experimental parameters such as solution
concentrations, bead surface chemistries, and the
order of protein binding events
3. the formation of large protein complexes could be
investigated using this technique
4. fluorescence detection and protein labeling will be
needed for studying small proteins using this
technique.
This project will continue into fiscal year 2001 to develop
methods for obtaining quantitative protein binding data
for biologically important protein systems, and also
incorporate on-column fluorescence detection to allow the
detection of small proteins.
References
Anti- Human IgG-TRITC
O.1M HCl
Figure 2. Automated protein binding experiments demonstrating the selective binding of multiple proteins onto a renewable
microcolumn. The system is shown schematically in (a) and on-column absorbance data at three wavelengths are shown in (b).
The column is perfused at 0.5 µL/s with three proteins (40 µg/mL, 50 µL). Each injection is followed by 150 µL phosphate buffered
saline (0.5M HCl, pH 7.4). Chicken IgG does not bind to the protein G Sepharose beads (demonstrates negligible nonspecific
protein binding), but subsequent injections of human IgG and goat anti-human IgG bind to the microcolumn. The protein binding
can be monitored in the UV region (due to aromatic amino acids) and also at the wavelengths of specific labels on the proteins
(fluorescein=496 nm and tetramethylrhodamine=557 nm).
280
(b)
496
557
Bruckner-Lea CJ, DA Holman, BL Schuck, FJ Brockman,
and DP Chandler. 1999. “Strategies for automated
sample preparation, nucleic acid purification, and
concentration of low target number nucleic acids in
environmental and food processing samples.” In SPIE
Proceedings 1999, 3544:63-71.
Biosciences and Biotechnology 43