Selex
SELEX: Systematic Evolution of Ligands via Exponential Selection is the process of identifying specific aptamers.
To select a binding aptamer, you don't look for epitopes. This only simplifies the process as you don't design a determined structure but reduced little by little an already binding population.
The SELEX screening process starts with a random library of nucleotide oligomers of a fixed size with know sequences in each end (for further amplification by PCR). Then the library is incubated with the target molecule. A number of this random sequences will bound to the target and the unbound sequences will be discharged.
The bound sequences are separated now from the target molecule (elution step) and amplified to create a new library.
With every round, more and more oligonucleotides with low binding-affinity for our protein of interest will be discarded leaving only strong binding aptamers at the end:
Our experiment:
To separate the bound sequences from the unbound ones, we decided to use a nitrocellulose membrane.
Nitrocellulose membranes can bind protein through hydrophobic interactions, so when applying the incubated library pool with the target protein to the membrane and forced the liquid to pass through it, the protein will stay within the membrane and as well the bound sequences.
The pore size was chosen after folding the aptamers and test both 0,22 um and 0.45 um size pore. After seeing the results we conclude that the 0.22 um pore was too small and the aptamers were being trapped because of their size and not by unspecifically interactions.
We tried in parallel two different proteins:
BSA
Ole-e1