Experiments with magnetic beads
For this project we used magnetic beads coated with Streptavidin, namely Dynabeads™ MyOne™ Streptavidin T1.
Streptavidin has an extremely high affinity for a water soluble B-vitamin called biotin (Vitamin B7), the dissociation constant, Kd, of these two substrates is around 10-15 M. Streptavidin's affinity for biotin is one of the strongest naturally occurring non-covalent bonds, which is why Streptavidin is a prime component in assays involving biotinylated substrates.
Furthermore, these beads are also magnetic, hence they can be pulled aside by a magnet to allowing the beads to be easily washed and re-suspended in different solutions. Therefore we were able to sequentially bind different molecules on top of one another on the surface of the beads, forming the basis of the pull-down assay.
A pull-down assay using magnetic beads serves as an accessible and rapid method that requires little equipment other than the beads, a system that can shake the beads at relatively low velocity during incubation periods and a system adapted measure the final readout of a given assay.
A pull-down assay consists in flowing various solutions containing the substrates that we want to bind to the surface of the streptavidin coated beads, incubating these solutions, removing them and then washing the beads to eliminate anything that is not bound to the beads directly or in a complex.
Double bind pull-down assay
Within the confines of this project, a double bind pull-down assay was performed to reproduce the initial epitope binding assay results achieved using the microfluidic platform. It first involves coating the beads with thrombin by re-suspending them in a solution of biotinylated thrombin. Then the beads are re-suspended in a solution containing two aptamers with different fluorescent labels (Thrombin aptamer 1 and 2) and the presence of each aptamer is detected using a plate-reader.
Although a double bind assay provided a good proof-of-concept, it is not applicable to situations outside of the laboratory, as the protein needs to be biotinylated.
A sandwich assay, as defined in this project, consists of binding a biotinylated aptamer on the surface of Streptavidin coated beads, followed by the binding of a target protein and finally another aptamer, which is labeled with a fluorescent marker to enable detection of the complex. Alternatively the second aptamer can be extended to include a trigger sequence that instigates the expression of a reporter upon the addition of cell lysate.
Sandwich assays, in contrast with double bind assays, can be used to detect naturally occurring substrates, since said substrates do not need to be biotinylated.