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MOLECULAR BIOLOGY: WORKING WITH DNA

PROTEIN EXPRESSION: LABELING

TCA Precipitation to Determine the Incorporation of Radiolabeled Amino Acids into Protein

TCA Precipitation to Determine the Incorporation of Radiolabeled Amino Acids into Protein
 
Overview
This protocol can be used to measure the amount of incorporation of 35S-Methionine or other radiolabeled amino acids into cellular protein following a metabolic labeling procedure. A protein extract needs to be made before commencing this protocol.
 
Procedure
1. Spot 1 to 2 μl of radiolabeled protein extract (CAUTION! see Hint #2) onto a small pieces of Whatman glass microfiber GF/C or hardened ashless 541 filter paper (see Hint #1).

2. Allow filters to dry at room temperature or under a heat lamp.

3. Add filter samples to about 60 ml of boiling 10% TCA (CAUTION! see Hint #2).

4. Boil for 5 min.

5. Wash the filters in ice-cold 5% TCA for 3 min.

6. Repeat the wash in ice-cold 5% TCA.

7. Wash the filters in ice-cold 95% Ethanol for 1 min.

8. Repeat the wash in ice-cold 95% Ethanol.

9. Allow the filter samples to dry.

10. Add the filters to the scintillation vial along with a toluene-based scintillation fluid.

11. Count the samples in a scintillation counter.

Solutions
95% (v/v) Ethanol
5% (v/v) TCA
10% (v/v) TCA
100% TCA Stock   Dissolve in 227 ml ddH2O
500 g Trichloroacetic Acid (TCA)
 
BioReagents and Chemicals
Scintillation Fluid
Trichloroacetic Acid
Ethanol
 
Protocol Hints
1. For larger sample volumes, increase the size of the filter paper.

2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.