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Proteins needed - CD79A
- normal
- fusion: CD79A C terminus + tev cut site + FP(mKATE)
- CD79B
- normal
- fusion: CD79B C terminus + tev cut site + FP(eBFP)
- Lyn
- normal
- fusion: FP tagged (N or C terminus?)
- point mutation: mutated to always phosphorylate
- Syk
- fusion, point mutation: FP attached(N or C terminus?), prevent phosphorylation (eYFP tagged SYK shouldn't be in same cell as FP anything else, ought be no cross talk)
- fusion, point mutatino: tev protease attached (N or C terminus?), point mutated to prevent phosphorylation
- ig k
- with proper localization tag
- igM heavy
- with proper constant region for membrane localization and proper localization tag
- dito + tev +eYFP
we have eBFP(2?), eYFP, mKATE
Supplies needed - Antibody that targets membrane bound igM that is dyed so we can see it (we can buy this, probably target heavy chain, may also want light chain as well to make sure both localize)
- Beta amyloid (? some kind that binds to cells and some kind we can wash antibodies over?)
- Lyn and Syk phosphorylation assay
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Things to test:
- does antibody stick to beta amyloid?
- Membrane localization
- does antibody get to membrane?
- does cd79A/B get to membrane?
- does lyn attach?
- does syk get recruited by lyn?
- ? test CD79x dimerization with gel
- ?test CD79x-IgM attachement with gel
- does TEV cut anything?
- does TEV affect Syk localization(assay for phosphorylation of syk)
- does TEV linked to syk cleave cut correct site?
- -how much does it cut as a function of amount of Syk?
- -where should we put molecule to be cleaved?
- -Does using a transcription factor work (drive an FP), do after finding where to attach
does beta amyloid trigger recruitment of sykdoes beta amyloid trigger release of signal- final: beta amyloid produces output
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