Initial Screening of CD79B-X-TCS-X-Gal4VP16 was largely uninformititve. The data did not appear to be statistically significant. We think this is likely due to minimal expression of the B-Cell Receptor ~5% drowning out our signal. To attempt to boost our signal, we are optimizing receptor expression relative to Syk-X-TEV/tTEV. Although our data appeared to be insignificant, we selected our three most promising candidates based on normalizing the fluorescent signal to our transfection marker. The linkers selected are S12 12TCS12, S9 9TCS15, and S6 12TCS6.
| Untransfected | Single Color Blue | Background anti-IgM binding | Single Color Red | Double Color | -VE control (background activation) | -VE control (background cleavage) | +VE control |
|---|
| HEK293 | HEK293 hEF1a:CymR hEF1a:rtTA CuO:MabH CuO:MabL hEF1a:Lyn hEF1a:eBFP CuO:CD79A-12TCS12-G4VP16 CuO:CD79B TRE:Syk-112-tTEVp
Dox 1 nM Cumate 500uM | HEK293 hEF1a:CymR hEF1a:rtTA CuO:MabH CuO:MabL hEF1a:Lyn hEF1a:eBFP CuO:CD79A-12TCS12-G4VP16 CuO:CD79B TRE:Syk-12-tTEVp
Dox 1 nM Cumate 500uM anti-IgM 1uL/100uL | HEK293 hEF1a:CymR hEF1a:rtTA CuO:MabH CuO:MabL hEF1a:Lyn hEF1a:mKate CuO:CD79A-12TCS12-G4VP16 CuO:CD79B TRE:Syk-12-tTEVp
Dox 1 nM | HEK293 hEF1a:CymR hEF1a:rtTA CuO:MabH CuO:MabL hEF1a:Lyn hEF1a:eBFP hEF1a:mKate CuO:CD79A-12TCS12-G4VP16 CuO:CD79B TRE:Syk-12-tTEVp
| HEK293 hEF1a:CymR hEF1a:rtTA CuO:MabH CuO:MabL hEF1a:Lyn hEF1a:eBFP UAS:mKate CuO:CD79A CuO:CD79B TRE:Syk-12-tTEVp
Dox 1 nM Cumate 500uM | HEK293 hEF1a:CymR hEF1a:rtTA CuO:MabH CuO:MabL hEF1a:Lyn hEF1a:eBFP UAS:mKate CuO:CD79A-12TCS12-G4VP16 CuO:CD79B TRE:Syk
Dox 1 nM Cumate 500uM | HEK293 hEF1a:CymR hEF1a:rtTA CuO:MabH CuO:MabL hEF1a:Lyn hEF1a:eBFP UAS:mKate CuO:CD79A CuO:CD79B TRE:Syk-12-tTEVp hEF1a:GAl4VP16
Dox 1 nM Cumate 500uM |
Plate Map
WELL 25 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:GMabH 50ng CuO:GMabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-9tcs15-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 0uM | WELL 26 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:GMabH 50ng CuO:GMabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-9tcs15-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 0uM anti IgM 1uL/100uL | WELL 27 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:GMabH 50ng CuO:GMabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-9tcs15-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 10uM | WELL 28 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:GMabH 50ng CuO:GMabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-9tcs15-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 10uM anti IgM 1uL/100uL | WELL 29 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-9tcs15-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 500uM | WELL 30 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-9tcs15-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 500uM anti IgM 1uL/100uL |
WELL 31 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs6-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 0uM | WELL 32 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs6-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 0uM anti IgM 1uL/100uL | WELL 33 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs6-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 10uM | WELL 34 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs6-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 10uM anti IgM 1uL/100uL | WELL 35 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs6-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 500uM | WELL 36 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs6-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 500uM anti IgM 1uL/100uL |
WELL 37 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs12-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 0uM | WELL 38 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs12-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 0uM anti IgM 1uL/100uL | WELL 39 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs12-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 10uM | WELL 40 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs12-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 10uM anti IgM 1uL/100uL | WELL 41 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs12-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 500uM | WELL 42 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:MabH 50ng CuO:MabL 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs12-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 500uM anti IgM 1uL/100uL |
WELL 49 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-9tcs15-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 0uM | WELL 50 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-9tcs15-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 0uM anti IgM 1uL/100uL | WELL 51 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-9tcs15-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 10uM | WELL 52 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-9tcs15-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 10uM anti IgM 1uL/100uL | WELL 53 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-9tcs15-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 500uM | WELL 54 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuOVRC01 L50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-9tcs15-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 500uM anti IgM 1uL/100uL |
WELL 55 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs6-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 0uM | WELL 56 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs6-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 0uM anti IgM 1uL/100uL | WELL 57 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs6-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 10uM | WELL 58 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs6-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 10uM anti IgM 1uL/100uL | WELL 59 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs6-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 500uM | WELL 60 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs6-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 500uM anti IgM 1uL/100uL |
WELL 61 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs12-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 0uM | WELL 62 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs12-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 0uM anti IgM 1uL/100uL | WELL 63 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs12-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 10uM | WELL 64 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs12-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 10uM anti IgM 1uL/100uL | WELL 65 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs12-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 500uM | WELL 66 HEK293 hEF1a:rtTA 50ng hEF1a:CymR 50ng CuO:VRC01 H 50ng CuO:VRC01 L 50ng hEF1a:Lyn 50ng hEF1a:eBFP 50ng UAS:mKate 50ng CuO:CD79A-12tcs12-Gal4VP16 50ng CuO:CD79B 50ng TRE:Syk-12-tTEVp 50ng
Dox 1 nM Cumate 500uM anti IgM 1uL/100uL
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