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group of experiments, we want to test
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whether or not the
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receptors, LilrB2 and PirB, localize to the
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cell membrane. We will test for membrane localization by immunostaining for LilrB2 and PirB and analyzing the cell population through both cytometry and microscopy.
Our cytometry analysis of the first trial of the membrane localization test didn't give us
Group B: Receptor Affinity for Beta-amyloid
In this
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group of experiments, we
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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
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oligomers to
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the cells expression the LilrB2 and PirB fusions. 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 fluorescent protein and the beta-amyloid.
Group C: Levels of Endogenous Cofilin
comparing endogenous Syk expression to expression of some protein under TRE, since in theory the expression level of TRE:(random protein) should reflect the amount of Syk-TEVp we'll get out from TRE:Syk-TEVp. What Brian suggested was that the "random protein" we use should actually be Syk-TEVp or Syk-eYFP (I guess it removes some of the variables). This way we can compare endogenous and exogenous Syk production directly. In a Western blot, this involves essentially using the antibodies to stain two different proteins - the native Syk and a Syk-fusion. Since Syk and Syk-fusion are different sizes, there should be two different bands on the Western blot afterwards, whose strength we can compare. I'm not sure whether we'll end up using Syk-eYFP or Syk-TEVp for our blots but I can't imagine that it matters all that much. (Maybe we'll try out both?)
Group D: Cofilin Recruitment
- Find data on receptor-cofilin dynamics (esp. how long cofilin stays recruited at the membrane)
- pEXPR TRE:cofilin-eYFP
- look for higher intensity at around membrane
- pEXPR hef1a:eBFP
In this experiment, we want to want to compare the levels of endogenous cofilin and cofilin-eYFP expressed under an inducible promoter with different levels of dox induction. This will allow us to determine what level of expression of cofilin would lead to the best signal:noise ratio of binding of exogenous cofilin. We are planning to transfect HEK293 with cofilin-eYFP under a TRE promoter and induce with varying levels of dox. We will then preform a Western blot with an anti-cofilin primary antibody and compare band sizes to test for the relative amounts of endogenous cofilin and exogenous cofilin-eYFP in the cells.
Group E: Testing TEVp-Cofilin cleavage with active receptors
In this experiment, we want to test for the recruitment of our engineered cofilin to the activated receptor proteins. To do this, we will transfect HEK293 cells with LilrB2 and PirB as well as coflin-eYFP under a TRE promoter. We will then apply beta-amyloid oligomers in order to activate the receptors and add varying amounts of dox to induce varying levels of expression of cofilin-eYFP. We will look for increased yellow light intensity near the membrane of the cell. We test different levels of expression of cofilin because we don't know how the levels of endogenous cofilin in the cell will affect the interaction between our receptor and our engineered cofilin. We are looking for the level of expression of cofilin that will result in a discernible increased intensity of yellow near the membrane, indicating that a significant amount of cofilin-eYFP has recruited to the membrane.
Group E: (Whole System) Cofilin + TEV Protease Recruitment to Receptor + Cleavage Site + Transcription Factor
- pEXPR hEF1a:Lilrb2-TCS-Gal4VP16
- pEXPR TRE:cofilin-TEV
- pEXPR Gal4UAS:eYFP
- pEXPR hEF1a:rtTA3
- pEXPR hEF1a:eBFP transfection marker
- pEXPR TRE:mKate indicator of effect of dox on transcription
How much TEV Protease we need in the system? - indicated by levels of dox
looking for biggest difference in transfer curve between w/ AB and without AB
two different ladders - dox and AB