The interaction of specific DNA sequences with proteins is central to many cellular processes including transcription, replication, repair, and gene regulation. The recognition sequence can be determined by a number of techniques, such as electrophoretic mobility shift assays (EMSA) and filter binding assays.
The technique of Surface Plasmon Resonance (SPR) is ideally suited as an alternative technique to measure these interactions because it is quantitative, simple to perform, reproducible, can be automated, and requires very little sample. RedcaT (Reusable DNA Capture Technology) uses SPR but allows a single chip to be reused multiple times so that many DNA sequences can be tested at low cost.
i) If you have a sequence which you believe is the binding site for your protein then this can be tested and compared with other sequences. For example, sequences from a MEME-predicted binding site.
ii) Identifying the binding sequence from a promoter region by breaking it into a number of overlapping sequences and comparing the interactions with your protein.
Our prices for the Redcat service are currently under review. Please enquire for more information.
There is a setup fee which covers the cost of setting up the assay for a new study. So if we have tested your protein and you subsequently decide to have more oligos tested then there will be no second setup fee for these, just the sample fees. We will also store your protein for a period of time, if requested, so that if you do decide to have more DNA sequences tested then you don’t have to send more protein.
A control run is made at the start and end of each set of samples to ensure that the process is working correctly. This uses a protein and DNA sequence which give a known response.
The sample is also run against the control DNA.
We provide a report containing the output from the interaction experiments and a brief analysis.
i) How much protein to send. This depends on the number of runs to be made but a minimum of 10μl at 100μM concentration.
ii) How to send the protein. The protein should be at a concentration of 100μM and sent on dry ice.
iii) Protein quality:-
You should ensure that your protein is soluble in conditions similar to the assay buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20). Your protein should also be filtered (0.22 μm filter). Please also send information about any small molecules included in the sample buffer (such as DMSO) as these will need to be incorporated into the running buffer.
iv) Description of the DNA sequences to be tested. The sequence can be up to 40 bases in length (we can perform the experiment with longer sequences but these are not standard lengths so please contact us). For each double-stranded DNA sequence to be tested please insert the complementary sequences (forward and reverse) into the order from attached (Redcat order form). You don’t need to add the single-stranded section for immobilization, we will do this.
Alternatively, and we recommend that you do this, you can download a copy of the program to generate overlapping oligos (PooP) using the link below. This program enables you to input your sequence of interest and then generate a series of overlapping DNA fragments to which you can attach the ssDNA linker required for the ReDCaT method. You can specify how long you wish each fragment to be and how many base pairs should be incorporated into the overlap.