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MOLECULAR BIOLOGY: WORKING WITH DNA

PROTEIN EXPRESSION: LABELING

NaOH-Peroxide Method to Determine the Incorporation of Radiolabeled Amino Acids into Protein

NaOH-Peroxide Method 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. Add 2 μl of radiolabeled protein extract to 500 μl ddH2O.

2. Add 250 μl Peroxide Solution.

3. Incubate for 20 min at 37°C.

4. Add 500 μl 25% TCA and 10 μl BSA.

5. Incubate on ice for 30 min.

6. Collect precipitate on Whatman glass microfiber GF/C filter discs using a vacuum manifold.

7. Wash the filters 3 times with 5 ml of 5% TCA.

8. Wash the filters 3 times with 5 ml of 95% Ethanol.

9. Air dry filters.

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

11. Count samples in a scintillation counter.

Solutions
5% (v/v) TCA Stock
25% (v/v) TCA Stock
25 mg/ml Bovine Serum Albumin (BSA)
100% TCA Stock   Dissolve in 227 ml ddH2O
500 g Trichloroacetic Acid (TCA)
Peroxide Solution   1 M NaOH
Make up just before use.
0.01% (w/v) unlabeled amino acids (as carrier protein)
2% Hydrogen Peroxide
 
BioReagents and Chemicals
Scintillation Fluid
BSA
Trichloroacetic Acid
Unlabeled Amino Acids
Ethanol
Hydrogen Peroxide
Sodium Hydroxide
GF/C filters
 
Protocol Hints
No hints are associated with this bioProtocol