Measurement of Incorporation of 35S-Cysteine into Cellular P

Sunday, October 19, 2003

Description
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 l of radiolabeled protein extract to 70 l of Urea Solution.

2. Incubate at 100C for 2 min.

3. Add 20 l 1 M Iodoacetic Acid.

4. Incubate at 37C for 30 min.

5. Add 2 l 2-mercaptoethanol.

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

7. Incubate at 100C 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 l Protosol.

12. Incubate at 55C for 1 hr.

13. Add 10 ml of scintillation fluid.

14. Count the samples in a scintillation counter.

Recipes
5% (v/v) TCA

10% (v/v) TCA

100% TCA Stock Dissolve in 227 ml ddH2O
500 g Trichloroacetic Acid (TCA)

1 M Iodoacetic Acid Prepare fresh

Urea Solution 0.5% (v/v) 2-mercaptoethanol
10 M Urea

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Measurement of Incorporation of 35S-Cysteine into Cellular P