Application Notes

4 which show that substitution of this residue results in a weaker affinity of BLIP to TEM1. A B Fig. 4: binding of Ypet-BLIP to TEM1 protein. In the MST experiment we kept the concentration ofYpet- BLIP-WT constant at 15 nM, while the concentration of TEM1 was varied. After 10 min an MST analysis was performed (n = 3). A) Binding of Ypet-BLIP-WT to TEM1-WT. A K d of 4.7 nM ± 0.75 nM was determined for this interaction. B) Binding of Ypet- BLIP-WT to TEM1-R243A. A K d of 250 nM ± 65 nM was determined for this interaction. Since biochemical processes take place in a highly crowded macromolecular environment in vivo, it is important to study interactions under conditions closer to those of the cells, such as in cell lysates. Additionally, using fluorescent fusion proteins to study binding in cell lysates is very convenient, since the fluorescent fusion protein do not need further purification. Thus time and the amounts of biological materials needed can be saved. Here, we have analyzed the binding of Ypet-BLIP-WT to TEM1-WT in mammalian cell lysates. First 12 dilutions of WT TEM1 were prepared in MST buffer. 15 nM BLIP was added to 293T cell lysate (20 million cells were lysed in 500 µl of RIPA-buffer). Then the cell lysate was centrifuged for 5 minutes at 15,000 rpm. 10 µl ofYpet-BLIP-containing lysate was added to each dilution of TEM-WT . After a short incubation, the samples were loaded into MST glass capillaries (K002, NanoTemper Technologies) and the analysis was performed. As expected, the K d which was determined was in the low nM range as it was for the determination performed MST buffer. Fig. 5: binding of Ypet-BLIP to TEM1 protein analyzed in 50 % mammalian cell lysate. TEM1 was titrated in 1:1 dilutions in MST-buffer. Then Ypet-BLIP was added into 200 µl of 293T cell lysate to a final concentration of 20 nM. 10 µl of BLIP-containing cell lysate was mixed with 10 µl TEM1 dilutions. For measurements, samples were filled into standard treated capillaries (n = 3). A K d of 5.7 nM ± 0.75 nM was determined for this interaction.

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