SLM Aminco 48000 MHF Fluorometer Setup

 

  1. Make sure all instrumentation is off before turning on lamp
  2. Add or remove emission monochromator as needed Ð make sure photomultiplier (detector) is attached
  3. Turn on lamp power source, wait 5 seconds and ignite lamp
  4. Turn on Modulator
  5. Set temperature and turn on water bath
  6. Turn on stirrer
  7. Turn on fluorometer
  8. Turn on computer and monitor
  9. Type Ò48000Ó
  10. Hit space to exercise monochromators
  11. Hit return if computer ÒCanÕt find dataÓ
  12. Data acquisition

Anisotropy

  1. Single point polarization for protein titrations (anisotropy)
  2. Load program (mine is ÒmatttamÓ)
  3. Hit ÒTÓ for turn turret
  4. Add filters (orange (cs-69) for TAMRA; yellow (cs-67) for fluoroscein)
  5. Add sample (1cm2 cuvette) to sample compartment
  6. Open lamp shutter (red knob up)
  7. A, C polarizer at 0; B polarizer at 9
  8. Adjust HV (high voltage) with shutters open Ð F6, then F9 or F10 Ð so that each channelÕs detector will stay within range, yet have enough signal (A,B~2-3 (may more than double over emperiment); C~4-5 (should not change over experiment))
  9. Hit enter to begin taking anisotropy readings
  10. Take 3 air blanks
  11. Take 6 anisotropy averages and record
  12. Hit Esc to record total intensity (A/C) at magic angle (ex polarizer at 0; em polarizer at 5)
  13. Repeat 23 (or 22) for three readings
  14. With all shutters (channels) closed, remove lid and add protein aliquot
  15. Let protein sample equilibrate 3 minutes, open shutters, and goto 21
  16. When done, turn of instruments in reverse order (lamp last)
  17. Yell at Matt or Joel for everything Matt forget to include in this protocol