observed. Alternatively, after the protein has bound to the DNA, a
ligand can be injected over the chip to assess whether the interaction is affected.
Most of the interactions studied to date using this method have
been protein:DNA interactions, but it is also possible to use the
method to study protein:RNA interactions. In this case, the test
RNA would have the DNA ReDCaT linker attached creating a
RNA:DNA hybrid for the reverse strand.
To summarize, the chip can be used hundreds of times, making
the SPR technique for testing protein:DNA interactions attractive.
Any protein or DNA sequence can be quickly tested using very
small quantities of protein. The results are highly reproducible, and
quantitative data can be obtained. Although the ReDCaT method
requires the use of an expensive SPR instrument, these are often
available through access to shared scientific services at many institutions, with experts available to assist users. Thus, the technique
should be considered as a complement to any study that analyzes
protein:DNA interactions, irrespective of the expertise of the
investigator.
4 Notes
1. A wide range of running buffers can be used, but it is important
to pick one that contains the ingredients necessary for the
interaction to occur. For example: if the interaction requires
the presence of magnesium, this should be added to the running buffer. The buffer given in the method is a good starting
point and, in our lab, has proved successful for a wide range of
different protein samples.
2. This method has been developed to be implemented using the
Biacore T200 SPR instrument and the Series S SA chip
(GE Healthcare). However, it could easily be adapted to run
with any SPR instrument and compatible streptavidin chip.
3. The protein should be purified and concentrated before dilution into the running buffer. For the first experiment, it is
recommended to use two protein concentrations (1 and
0.1 μM). For subsequent experiments, a range of concentrations may be tested. The protein concentration should be
calculated assuming the protein is monomeric.
4. The ReDCaT linker used is 20 nucleotides long and is designed
to have no secondary structure or any tendency to anneal with
itself, as assessed by the Sigma-Aldrich website (www.sigmagonosys.com/calc/DNACalc.asp). If there is any suspicion
that the protein binds to the ReDCaT linker, this should be
tested using just the linker or a randomized sequence plus
linker. If the linker is found to contain a specific binding site,
376
Clare E. M. Stevenson and David M. Lawson
ligand can be injected over the chip to assess whether the interaction is affected.
Most of the interactions studied to date using this method have
been protein:DNA interactions, but it is also possible to use the
method to study protein:RNA interactions. In this case, the test
RNA would have the DNA ReDCaT linker attached creating a
RNA:DNA hybrid for the reverse strand.
To summarize, the chip can be used hundreds of times, making
the SPR technique for testing protein:DNA interactions attractive.
Any protein or DNA sequence can be quickly tested using very
small quantities of protein. The results are highly reproducible, and
quantitative data can be obtained. Although the ReDCaT method
requires the use of an expensive SPR instrument, these are often
available through access to shared scientific services at many institutions, with experts available to assist users. Thus, the technique
should be considered as a complement to any study that analyzes
protein:DNA interactions, irrespective of the expertise of the
investigator.
4 Notes
1. A wide range of running buffers can be used, but it is important
to pick one that contains the ingredients necessary for the
interaction to occur. For example: if the interaction requires
the presence of magnesium, this should be added to the running buffer. The buffer given in the method is a good starting
point and, in our lab, has proved successful for a wide range of
different protein samples.
2. This method has been developed to be implemented using the
Biacore T200 SPR instrument and the Series S SA chip
(GE Healthcare). However, it could easily be adapted to run
with any SPR instrument and compatible streptavidin chip.
3. The protein should be purified and concentrated before dilution into the running buffer. For the first experiment, it is
recommended to use two protein concentrations (1 and
0.1 μM). For subsequent experiments, a range of concentrations may be tested. The protein concentration should be
calculated assuming the protein is monomeric.
4. The ReDCaT linker used is 20 nucleotides long and is designed
to have no secondary structure or any tendency to anneal with
itself, as assessed by the Sigma-Aldrich website (www.sigmagonosys.com/calc/DNACalc.asp). If there is any suspicion
that the protein binds to the ReDCaT linker, this should be
tested using just the linker or a randomized sequence plus
linker. If the linker is found to contain a specific binding site,
376
Clare E. M. Stevenson and David M. Lawson