Wittrup: Fluorescein Quench Titration Assay
1.5 Fluorescein Quench Titration Assay
(adapted from Katerina Midelfort’s Thesis)
1. Express 4m5.3 through growth in SG-CAA or YPG. Collect supernatant by spinning down the cells.
2. Collect 0.5 mL supernatant for the assay. 3. Add sample to 2.5 mL Assay Buffer, TBS (50 mM Tris, 150 mM NaCL pH 8.0)
4. Place in cuvette holder in Fluorescence Spectrophotometer Settings:
a. Excitation: 494 nm, slit 2.5, filter auto
b. Emission: 517, slit 5.0, filter open (sometimes use a OG495 Melles Griot yellow filter to keep excitation light from scattering into the detector.)
c. PMT: medium, average time: 0.2 seconds with 3 samples taken at each point.
d. Temperature control set to 30°C
5. Take initial reading.
6. Add fluorescein (most experiments use 10 μM fluorescein dissolved in TBS). I usually start by adding in 3 μl increments. Wait five minutes and take a reading. Wait an additional five minutes and take another reading. Keep waiting and measuring until the same absorbance is reached for two readings in a row. Then, add more fluorescein.
7. Repeat this process until the increase in signal with each addition is similar to the increase seen for a negative control of supernatant lacking any 4m5.3.
8. Correct for the background and determine and derive a calibration curve by doing the experiment in supernatant without fluorescein (as described above.)
9. Fit the data to the expression: where F is fluorescein concentration in the cuvette, M is 4m5.3 concentration in the cuvette, q is the quench constant (0.95 for 4m5.3), and the Kd for 4m5.3 is 0.3 pM for 4m5.3. c is the slope of the line from the calibration curve.
10. You can find the 4m5.3 concentration by fitting that expression to the data and finding a M value that gives the best fit.
Andy Rakestraw May 5, 2006