out. This can be selected when the method is run. For all experiments, tubes should be loaded with the appropriate amount of
solution and placed in the rack as detailed in the Biacore T200
Control Software.
3.1 Preparation
of the ReDCaT Chip
1. To remove any unconjugated streptavidin, both flow cells
(FC ref and FC test ) are washed using three injections of 1.0 M
NaCl, 50 mM NaOH for 60 s, each followed by buffer for 60 s
(all at 10 μl/min).
2. Then inject the ReDCaT linker (100 nM) at 5 μl/min over flow
cell 2 (FC test ) only to give a relatively high immobilization level
(approx. 500 RU) (see Note 8). This biotinylated ssDNA ReDCaT linker is now permanently immobilized via the streptavidin, thereby creating the ReDCaT chip. This chip can be used
repeatedly for many experiments and can be stored (when not
in use) in running buffer at 4
C. We have found that the
ReDCaT chips will keep for over 1 year and still be functional.
3.2 A General
Protocol to Screen
for Protein:DNA
Interactions
The flow rate is set at 30 μl/min unless stated otherwise.
1. Inject test DNA (1.0 μM) over flow cell 2 (FC test ) at a flow rate
of 10 μl/min for 60 s, followed by buffer for 60 s.
2. Then inject the test protein at the required concentration
(or buffer-only control) over both flow cells (FC ref and FC test )
for 60 s, followed by buffer for 60 s.
3. If required, inject a solution of 0.5 M NaCl for 60 s over both
flow cells (FC ref and FC test ), followed by buffer for 60 s. This
step is optional, but is useful to examine the effect of NaCl on
the protein–DNA interaction (see Note 9).
4. Inject regeneration solution over both flow cells (FC ref and
FC test ) at a flow rate of 10 μl/min to remove the test DNA
(together with any remaining bound protein) to leave only the
ReDCaT linker bound to the chip. The response should return
to the level prior to step 1 (see Fig. 1b). Then flow buffer over
both flow cells for a further 60 s.
Steps 1–4 can be repeated as many times as required (e.g., with
different DNA and protein samples), in an automated fashion
without any further user intervention. This is called a “cycle” and
each cycle takes approximately 15 min. Ideally the first cycle should
be a conditioning cycle where DNA is captured on the second flow
cell (FC test ) and test protein is run over both flow cells and then
both flow cells are regenerated. It is also recommended to run a
negative control, which consists of a piece of DNA at the same
length as the test DNA, but with a randomized sequence (see Note
10). All samples should be tested at least twice and at more than
one protein concentration, and buffer-only samples should be used
SPR Analysis of Protein-DNA Interactions
373
solution and placed in the rack as detailed in the Biacore T200
Control Software.
3.1 Preparation
of the ReDCaT Chip
1. To remove any unconjugated streptavidin, both flow cells
(FC ref and FC test ) are washed using three injections of 1.0 M
NaCl, 50 mM NaOH for 60 s, each followed by buffer for 60 s
(all at 10 μl/min).
2. Then inject the ReDCaT linker (100 nM) at 5 μl/min over flow
cell 2 (FC test ) only to give a relatively high immobilization level
(approx. 500 RU) (see Note 8). This biotinylated ssDNA ReDCaT linker is now permanently immobilized via the streptavidin, thereby creating the ReDCaT chip. This chip can be used
repeatedly for many experiments and can be stored (when not
in use) in running buffer at 4
C. We have found that the
ReDCaT chips will keep for over 1 year and still be functional.
3.2 A General
Protocol to Screen
for Protein:DNA
Interactions
The flow rate is set at 30 μl/min unless stated otherwise.
1. Inject test DNA (1.0 μM) over flow cell 2 (FC test ) at a flow rate
of 10 μl/min for 60 s, followed by buffer for 60 s.
2. Then inject the test protein at the required concentration
(or buffer-only control) over both flow cells (FC ref and FC test )
for 60 s, followed by buffer for 60 s.
3. If required, inject a solution of 0.5 M NaCl for 60 s over both
flow cells (FC ref and FC test ), followed by buffer for 60 s. This
step is optional, but is useful to examine the effect of NaCl on
the protein–DNA interaction (see Note 9).
4. Inject regeneration solution over both flow cells (FC ref and
FC test ) at a flow rate of 10 μl/min to remove the test DNA
(together with any remaining bound protein) to leave only the
ReDCaT linker bound to the chip. The response should return
to the level prior to step 1 (see Fig. 1b). Then flow buffer over
both flow cells for a further 60 s.
Steps 1–4 can be repeated as many times as required (e.g., with
different DNA and protein samples), in an automated fashion
without any further user intervention. This is called a “cycle” and
each cycle takes approximately 15 min. Ideally the first cycle should
be a conditioning cycle where DNA is captured on the second flow
cell (FC test ) and test protein is run over both flow cells and then
both flow cells are regenerated. It is also recommended to run a
negative control, which consists of a piece of DNA at the same
length as the test DNA, but with a randomized sequence (see Note
10). All samples should be tested at least twice and at more than
one protein concentration, and buffer-only samples should be used
SPR Analysis of Protein-DNA Interactions
373