Labeling Efficiency Measurement by TCA counting

Materials

  1. Labeled cell lysate
  2. 10% TCA: Dilute 100% TCA (4oC) at 1:10 with MilliQ H2O, put on ice
  3. Acetone, put on ice
  4. Eppendorf tubes
  5. Gilson pippette and tips
  6. CF/A filters and apparatus
  7. Tweezers
  8. Scintilation counter machine vials
  9. Ice

Procedure

  1. Pippet 5 ul labeled lysate into 1.5ml tube
  2. Add 100ul 10% TCA solution containing casin amino acids and incubate on ice for 15min.
  3. Place CF/A filters on unit and wet with 500ul ice cold 10% TCA.
  4. Turn the vacum on.
  5. Put 100ul lysate/TCA mixture on the center of the filter.
  6. Wash twice by adding 1ml ice cold 10% TCA.
  7. Wash by adding 1ml ice cold 100% ice cold acetone.
  8. Put filter membrane in scintilation vial using tweezers.
  9. Let vial open dry 10min at room temperature.
  10. Add 2-4ml scint fluid. Cap.
  11. Count in scintilation counting machine. (Instructions is in the drawer).

 

 

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