a different linker sequence and/or length could be used, but
this would need to be optimized with positive and negative
controls. However, this ReDCaT linker has been used successfully to test a wide range of different protein samples without
any such problems.
5. The test DNA can be of any length. However, we have found
that sequences of 20–40 nucleotides in length tend to work
well and are cheap to order. Ideally the sequence needs to be
long enough to include the whole binding site. If the DNA site
is not known and a longer segment of DNA requires testing to
locate a binding site, this can be easily fragmented into shorter
segments for testing. This is discussed extensively in [7]. The
reverse stand is always 20 nucleotides longer as it contains the
additional nucleotides to hybridize to the ReDCaT linker
attached to the chip.
6. A slight excess of the forward strand is used to ensure all the
DNA that binds to the chip will be dsDNA (since the F strand
does not contain the additional complement to the ReDCaT
linker, it will not bind to the chip surface).
7. In this example, the reference flow cell is kept blank. This is the
simplest protocol, and it will reveal any nonspecific binding of
the protein to the chip. However, there are other options,
including placing the ReDCaT linker (with or without its
complement sequence, but no test DNA) on the reference
flow cell, or even to immobilize a randomized test DNA
sequence via the ReDCaT linker.
8. In the Biacore control software, the user can request that the
immobilization step should aim for a required response. This is
particularly useful as the instrument will estimate the length of
injection time to achieve the required response. If this option is
unavailable, multiple short injections should be carried out
until the required immobilization level is achieved.
9. Proteins that bind weakly to DNA will be more easily removed
than those that bind more tightly by the 0.5 M NaCl wash. If a
ligand or inhibitor is known to interrupt the interaction, this
could be used instead. It is possible to do the same experiment,
but with different ligands or inhibitors as a regeneration solution, to test the effect of these on the interaction.
10. It is important to use a negative control in any analysis. Routinely, one of the test DNA sequences is randomized and used
as the negative control. This should be the same length as the
test sequence. There are a variety of online tools that will
randomize a DNA sequence, such as: http://www.bioinformat
ics.org/sms2/shuffle_dna.html
SPR Analysis of Protein-DNA Interactions
377
this would need to be optimized with positive and negative
controls. However, this ReDCaT linker has been used successfully to test a wide range of different protein samples without
any such problems.
5. The test DNA can be of any length. However, we have found
that sequences of 20–40 nucleotides in length tend to work
well and are cheap to order. Ideally the sequence needs to be
long enough to include the whole binding site. If the DNA site
is not known and a longer segment of DNA requires testing to
locate a binding site, this can be easily fragmented into shorter
segments for testing. This is discussed extensively in [7]. The
reverse stand is always 20 nucleotides longer as it contains the
additional nucleotides to hybridize to the ReDCaT linker
attached to the chip.
6. A slight excess of the forward strand is used to ensure all the
DNA that binds to the chip will be dsDNA (since the F strand
does not contain the additional complement to the ReDCaT
linker, it will not bind to the chip surface).
7. In this example, the reference flow cell is kept blank. This is the
simplest protocol, and it will reveal any nonspecific binding of
the protein to the chip. However, there are other options,
including placing the ReDCaT linker (with or without its
complement sequence, but no test DNA) on the reference
flow cell, or even to immobilize a randomized test DNA
sequence via the ReDCaT linker.
8. In the Biacore control software, the user can request that the
immobilization step should aim for a required response. This is
particularly useful as the instrument will estimate the length of
injection time to achieve the required response. If this option is
unavailable, multiple short injections should be carried out
until the required immobilization level is achieved.
9. Proteins that bind weakly to DNA will be more easily removed
than those that bind more tightly by the 0.5 M NaCl wash. If a
ligand or inhibitor is known to interrupt the interaction, this
could be used instead. It is possible to do the same experiment,
but with different ligands or inhibitors as a regeneration solution, to test the effect of these on the interaction.
10. It is important to use a negative control in any analysis. Routinely, one of the test DNA sequences is randomized and used
as the negative control. This should be the same length as the
test sequence. There are a variety of online tools that will
randomize a DNA sequence, such as: http://www.bioinformat
ics.org/sms2/shuffle_dna.html
SPR Analysis of Protein-DNA Interactions
377