Description:
In this group of experiments, we are aiming to test the affinity of our receptors, LilrB2 and PirB to beta-amyloid oligomers. To do that, we will have HEK293 cells express LilrB2 and PirB fused with YFP. We will then apply biotinylated beta-amyloid oligomers to the LilrB2 and PirB expressing cells. To test for binding, we are going to use the biotin-binding protein streptavidin covalently bonded to a fluorophore. We will look for co-localization of the fluorophore and the beta-amyloid.
*We may potentially extend this experiment to trying to characterize the interaction between beta-amyloid and the receptors i.e. try to generate saturation plots and calculate dissociation constants.
Cloning
Description:
Make HEK293 expressing LilrB/PirB - YFP fusions. We will test binding beta-amyloid in Experiment B2
Plan:
Status:
- PCRed extensions onto LilrB2 and PirB
- gel run to verify PCR products
- LilrB2 did not give expected band size
- why?
- PirB did not give expected band size
- pENTR used as template was not right
- LilrB2 did not give expected band size
- gel run to verify PCR products
- PCRed extensions onto YFP
- verification gel to be run
- After struggle bussing through a few PCRs (and then realizing that we were doing PCRs suboptimally), Brian did our PCRs
- We seem to have the right PCR products
Experiment
Description:
Testing beta-amyloid binding to receptor proteins using biotinylated oligomerized beta-amyloid. We will test for binding using he biotin-binding protein streptavidin covalently bonded to a fluorophore. We will look for co-localization of the fluorophore and the beta-amyloid.
Status:
Not yet started.