EHS Center - Protein Interactions and Proteomics Facility Core




			Proteomics Facility Core Fluorescence Polarization (FP) 1. Description: FP is proportional to the rotational velocity of a fluorescent species in solution. Rotational velocity is proportional to the size of the molecule so binding or cleavage events that change the size of the fluorescent species can be detected. 2. Application: Binding of fluorescent molecules under 10,000 mol wt. to larger molecules Release of a fluorescent molecule under 10,000 mol wt from a larger molecule 3. Limitations: The fluorescent molecule must be relatively small. Preferred size is <10 kd upper limit is < 20 kd The species the fluorescent molecule is binding to or released from must be relatively large 4. Advantages: It is a “homogeneous” assay so the binding is detected without a separation step Time courses can be followed in a single sample Titrations can be performed in a single sample It requires little technical skill and works in a high percentage of applications attempted 5. Equipment: PanVera Beacon FP System: A single tube reader, requires 1.2 ml of sample Tecan Polarian Microplate Reader: Accepts 96 and 384 well plates with volumes as low as 100 µl 6. Services: Consultation on designing FP experiments Training to use the equipment Establish parameters in the Polarian for the Fl-probe employed Label peptides or proteins with Fl-molecules HPLC purification of fluorescently labeled peptides and proteins HPLC and mass-spec based evaluation of fluorescently labeled peptides and proteins HPLC based analytical evaluation of peptides