once the biotinylated DNA has been captured on the chip surface,
the streptavidin–biotin interaction is so tight that it cannot be
removed without damage to the chip. In addition, if the protein
of interest binds tightly to the DNA, it may not be possible to
remove the protein without damage to the bound DNA or to the
chip surface. In practice, this means a new chip would be required
to study each protein or DNA combination, thereby making this
approach very costly.
To enable the routine and cost-effective use of SPR for the
study of any protein-oligonucleotide pair, previous work [4–6] was
extended, and this chapter will describe an indirect capture method
that uses a ReDCaT chip [7]. This ReDCaT chip can be repeatedly
used; any DNA sequence can be bound and then removed without
compromising the chip, allowing multiple samples to be tested in a
high throughput and automated manner using a single chip. The
method can be used not only to identify binding events but also to
obtain quantitative affinity and kinetic data [7, 8]. This chapter will
describe, with technical details, how this method is implemented
and how the results are analyzed.
2 Materials
2.1 Buffers
and Reagents
Prepare all solutions using ultrapure water and analytical grade
reagents. Prepare and store at room temperature and follow all
waste disposal regulations (see Note 1).
1. Running buffer: 10 mM HEPES, pH 7.4, 150 mM NaCl,
3 mM Ethylenediaminetetraacetic acid (EDTA), 0.05% (v/v)
Tween 20. Weigh 2.383 g HEPES (238.3 g/mol), 8.766 g
NaCl (58.4 g/mol), 0.877 g EDTA (292.2 g/mol) and add to
a graduated glass beaker. Add water to a volume of approximately 750 ml and then add 0.5 ml Tween 20. Mix and adjust
pH to 7.4 using 5 M NaOH then make up to 1000 ml with
water.
2. 0.5 M NaCl: Weigh 0.292 g NaCl and make up to 10 ml with
water.
3. Regeneration solution: 1 M NaCl and 50 mM NaOH. Weigh
5.844 g NaCl and 0.2 g NaOH pellets (40.0 g/mol) and make
up to 100 ml with water.
2.2 Instrument
and Chip and Protein
Sample (See Note 2)
1. Instrument and Chip: SPR Biacore T200 (GE Healthcare) and
Series S SA chip (GE Healthcare).
2. Protein samples to be tested: Dilute the purified protein samples in running buffer to 1.0 and 0.1 μM concentration (see
Note 3). For an initial binding check in duplicate, 100 μl of
each concentration will be sufficient.
370
Clare E. M. Stevenson and David M. Lawson
the streptavidin–biotin interaction is so tight that it cannot be
removed without damage to the chip. In addition, if the protein
of interest binds tightly to the DNA, it may not be possible to
remove the protein without damage to the bound DNA or to the
chip surface. In practice, this means a new chip would be required
to study each protein or DNA combination, thereby making this
approach very costly.
To enable the routine and cost-effective use of SPR for the
study of any protein-oligonucleotide pair, previous work [4–6] was
extended, and this chapter will describe an indirect capture method
that uses a ReDCaT chip [7]. This ReDCaT chip can be repeatedly
used; any DNA sequence can be bound and then removed without
compromising the chip, allowing multiple samples to be tested in a
high throughput and automated manner using a single chip. The
method can be used not only to identify binding events but also to
obtain quantitative affinity and kinetic data [7, 8]. This chapter will
describe, with technical details, how this method is implemented
and how the results are analyzed.
2 Materials
2.1 Buffers
and Reagents
Prepare all solutions using ultrapure water and analytical grade
reagents. Prepare and store at room temperature and follow all
waste disposal regulations (see Note 1).
1. Running buffer: 10 mM HEPES, pH 7.4, 150 mM NaCl,
3 mM Ethylenediaminetetraacetic acid (EDTA), 0.05% (v/v)
Tween 20. Weigh 2.383 g HEPES (238.3 g/mol), 8.766 g
NaCl (58.4 g/mol), 0.877 g EDTA (292.2 g/mol) and add to
a graduated glass beaker. Add water to a volume of approximately 750 ml and then add 0.5 ml Tween 20. Mix and adjust
pH to 7.4 using 5 M NaOH then make up to 1000 ml with
water.
2. 0.5 M NaCl: Weigh 0.292 g NaCl and make up to 10 ml with
water.
3. Regeneration solution: 1 M NaCl and 50 mM NaOH. Weigh
5.844 g NaCl and 0.2 g NaOH pellets (40.0 g/mol) and make
up to 100 ml with water.
2.2 Instrument
and Chip and Protein
Sample (See Note 2)
1. Instrument and Chip: SPR Biacore T200 (GE Healthcare) and
Series S SA chip (GE Healthcare).
2. Protein samples to be tested: Dilute the purified protein samples in running buffer to 1.0 and 0.1 μM concentration (see
Note 3). For an initial binding check in duplicate, 100 μl of
each concentration will be sufficient.
370
Clare E. M. Stevenson and David M. Lawson