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What is the fundamental difference between FRET and TR-FRET? (1 point)

FRET techniques do not usually have a high signal-to-noise ratio due to various contaminating/interfering signals. Can you identify 2-3 factors that contribute to this interference? (2 points).

Calpains are proteases that play a variety of roles in intracellular calcium signaling by catalyzing the cleavage of a wide range of protein substrates. If you were requested to set up an assay to monitor the cleavage of the synthetic substrate by the purified calpain enzyme, what kind of assay would you set up? If you had to generate or make reagents to set up the assay, please indicate them.

Describe in steps the assay design (4 points)

Types of controls you will need to show specificity, sensitivity, and selectivity of the assay (3 points

4. Vascular endothelial growth factor receptor 2 (VEGFR-2) is an endothelial cell-specific receptor that has inherent tyrosine kinase activity. The binding of the Vascular endothelial growth factor (VEGF) ligand to its receptor plays an important role in angiogenesis, a process by which new capillaries are formed. Enhanced angiogenesis is associated with a number of diseases including cancer. Your goal is to develop a homogeneous in vitro TR-FRET kinase assay for VEGFR-2 (purified protein) in an attempt to identify small molecules that inhibit kinase activity and in turn modulate angiogenesis.

Based on this background, please answer the following questions (15 points):

If you wanted to measure the kinase activity of VEGFR-2, what are some of the conventional methods/assays you would use? (2 points)

How would you apply a TR-FRET assay to measure the in vitro kinase activity of VEGFR-2? Describe in steps the assay design and potential readout outcome. (4 points)

While setting up your experiment, you accidentally discovered autophophorylation of receptor. What observation/results led you to believe that there was autophosphorylation of the receptor? (3 points)

What is the advantage of measuring autophosphorylation of the receptor rather than phosphorylation of the substrate? (2 points)

How would you determine robustness of the assay and validate the assay? (4 points)

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