Protocols

Measurement of Incorporation of 35S-Cysteine into Cellular Protein

Posted on 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 ìl of radiolabeled protein extract to 70 ìl of Urea Solution.

2. Incubate at 100°C for 2 min.

3. Add 20 ìl 1 M Iodoacetic Acid.

4. Incubate at 37°C for 30 min.

5. Add 2 ìl 2-mercaptoethanol.

6. Transfer 10 ìl 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 ìl Protosol.

12. Incubate at 55°C 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


Supplies


Tips


Submitted by: manny440