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In this experiment, we want to test that beta-amyloid binds to the receptor proteins that we have introduced to the cell. 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.
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?)
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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.
- 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
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