With approximately two weeks left, the main goal will be detecting binding of our new adaptamer constructs to both streptavidin and thrombin simultaneously. Presumably once we have a quanitifiable assay in which we can detect how much thrombin is binding There are a variety of ways to show this, outlined below:
- Gel shift: While it would be ideal to see a shift from pure adaptamer to pure adaptamer + thrombin and greater shift for adaptamer + thrombin + streptavidin in a gel, our struggles in the early weeks with this assay suggest that this will be futile.
- Biacore: Used by Tahiri-Alaoui et al., this fancy system seems to be convenient and quantitative in its characterization of binding interactions, but difficult to use when just beginning. Due to time constraints and the uncertain advantage of using it, we will put it aside for now.
- Bead assays: By far the most promising assay as we have had clear evidence of both aptamers and adaptamers binding to streptavidin and thrombin (separately). Unfortunately, initial results from simply running elutions from incubations of streptavidin beads with adaptamers and free thrombin showed no thrombin in any lane. Therefore, there are a few modifications on the procedures that we can try:
- Western blot
- Fluorescent detection of thrombin
- Solution viscocity change/ clumping
- Agarose/magnetic beads
- Polystyrene beads
With these in mind, here's the plan for the next couple weeks:
- Show that the linkaptamers of the v.2 adaptamers can come together through a gel.
- Try Western Blot with Cyano group.
- Rerun yesterday's gel and restain with SYBR gold.
- Test the ability of the streptavidin magnetic beads' binding to v.2 adaptamers.
- Show that A15, A35 can bind thrombin and streptavidin agarose beads separately; this should be easy to do and should be a pretty good indicator that the A20 and A50 constructs will also bind thrombin and streptavidin beads separately. Additionally incubate with thrombin; results will be used in preparation for 8/18 Western Blot.
- Depending on 8/16 results, try coupling thrombin to polystyrene beads using new or old procedure.
- Run Western blot using 8/17 experiment results.
- Depending on time available, incubate streptavidin beads + A35 + anti-thrombin antibody + secondary antibody; wash and check for fluoresence vs. control.
- If new oligos are in, check visocity from incubating streptavidin agarose/magnetic streptavidin beads with new oligos.
- Depending on previous results, pursue whatever is working.