in vitro spectroscopy

  1. 2x106 293T cells grown on 10 cm-diameter dish.
  2. Transfect 10 g of pRaichu with calcium precipitation method.
  3. After 4 to 6 hours, change medium with phenol red-free MEM with 10% FBS.
  4. Thirty-six hours after transfection, wash cells twice with PBS.
  5. Add 500 l of lysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.5% Triton X-100) .
  6. Scrape cells and take into microtube.
  7. Centrifugation for 15 min at 4 oC.
  8. Take sup to a new microtube. Donft be greedy. Contamination of cell debris will disturb the spectrograph.
  9. Add 1 ml of PBS.
  10. Analysis with fluorescent spectroscope. We use 3 ml cuvette. Put a stuffer in the bottom of cuvette, so that you can reduce the sample volume. Fluorescence was monitored by use of an excitation wavelength of 433 nm and scan fluorescence from 450 nm to 550 nm.