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MOLECULAR BIOLOGY: WORKING WITH DNA

PROTEIN EXPRESSION: LABELING

Measurement of Incorporation of 35S-Cysteine into Cellular Protein

Measurement of Incorporation of 35S-Cysteine into Cellular Protein
 
Overview
This protocol can be used to measure the amount of incorporation of 35S-Cysteine into cellular protein following a metabolic labeling procedure. This protocol reduces the amount of non-specific counts, which can be considerable with labeled Cysteine. A protein extract needs to be made before commencing this protocol.
 
Procedure
1. Add 10 μl of radiolabeled protein extract to 70 μl of Urea Solution.

2. Incubate at 100°C for 2 min.

3. Add 20 μl 1 M Iodoacetic Acid.

4. Incubate at 37°C for 30 min.

5. Add 2 μl 2-mercaptoethanol.

6. Transfer 10 μl to a new microfuge tube and add 1 ml 10% TCA.

7. Incubate at 100°C for 10 min.

8. Collect precipitate onto Whatman glass microfiber GF/C filters using a vacuum manifold.

9. Wash the filters 3 times with 5 ml 5% TCA.

10. Transfer filters to scintillation vials.

11. Add 500 μl Protosol.

12. Incubate at 55°C for 1 hr.

13. Add 10 ml of scintillation fluid.

14. Count the samples in a scintillation counter.

Solutions
1 M Iodoacetic Acid   Prepare fresh
Urea Solution   0.5% (v/v) 2-mercaptoethanol
10 M Urea
5% (v/v) TCA
10% (v/v) TCA
100% TCA Stock   Dissolve in 227 ml ddH2O
500 g Trichloroacetic Acid (TCA)
 
BioReagents and Chemicals
Urea
2-Mercaptoethanol
Trichloroacetic Acid
Protosol
Iodoacetic Acid
Scintillation Fluid
GF/C filters
 
Protocol Hints
No hints are associated with this bioProtocol