Background:
The literature is unclear on whether Lyn is always associated with the BCR complex or if it binds only upon BCR dimerization. We know from the literature that Syk will bind but only in the presence of Lyn or two other Src family kinases that are known to associate with the BCR. Lyn is known to be expressed by HEK 293 cells so there may be competition with the binding of our engineered versions of syk-TEV and lyn-TEV. The purpose of this experiment is to determine whether or not Lyn will function as a cofactor in the TANGO system with the BCR. Essentially we want to know if Lyn is always associated with the BCR or if it is recruited upon activation of the receptor. We will be using Gmab because we have not yet ordered or constructed any other antibodies.
Parts Needed:
- Gmab H
- Gmab L
- Syk-TEV
- Lyn-TEV
- Lyn
- CD79A
- CD79B
- CD79A-TCS-G4
- CD79B-TCS-G4
- UAS: mKate
- hEF1a: BFP
- abeta
- hEF1a: G4
Procedure:
| HEK control | eBFP 100 | BCR 100 Lyn 100 Syk-TEV 100 U:mKate 100 H:Gal4 100 eBFP 100 | BCR 100 Lyn 100 Syk-TEV 100 U:mKate 100 eBFP 100 Dummy 100 | ||
BCR-G4 100 h:eBFP 100 h:rtTA 100 H:Lyn 100 T:Syk-TEV 100 U:mKate 100 10 nM dox aIgM | BCR-G4 25 h:eBFP 100 h:rtTA 100 H:Lyn 100 T:Syk-TEV 100 U:mKate 100 10 nM dox aIgM | BCR-G4 6.25 h:eBFP 100 h:rtTA 100 H:Lyn 100 T:Syk-TEV 100 U:mKate 100 10 nM dox aIgM | BCR-G4 100 h:eBFP 100 h:rtTA 100 H:Lyn 100 T:Syk-TEV 100 U:mKate 100 10 nM dox | BCR-G4 25 h:eBFP 100 h:rtTA 100 H:Lyn 100 T:Syk-TEV 100 U:mKate 100 10 nM dox | BCR-G4 6.25 h:eBFP 100 h:rtTA 100 H:Lyn 100 T:Syk-TEV 100 U:mKate 100 10 nM dox |
BCR-G4 100 h:eBFP 100 h:rtTA 100 T:Lyn-TEV 100 U:mKate 100 Dummy 100 10 nM dox aIgM | BCR-G4 25 h:eBFP 100 h:rtTA 100 T:Lyn-TEV 100 U:mKate 100 Dummy 100 10 nM dox aIgM | BCR-G4 6.25 h:eBFP 100 h:rtTA 100 T:Lyn-TEV 100 U:mKate 100 Dummy 100 10 nM dox aIgM | BCR-G4 100 h:eBFP 100 h:rtTA 100 T:Lyn-TEV 100 U:mKate 100 Dummy 100 10 nM dox | BCR-G4 25 h:eBFP 100 h:rtTA 100 T:Lyn-TEV 100 U:mKate 100 Dummy 100 10 nM dox | BCR-G4 6.25 h:eBFP 100 h:rtTA 100 T:Lyn-TEV 100 U:mKate 100 Dummy 100 10 nM dox |
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Results:
Discussion: