Home | Achievement | Programmes | Projects | Experts | Staffs | Publications | Journals |
Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |

MOLECULAR BIOLOGY: WORKING WITH DNA

PROTEIN EXPRESSION: LABELING

35S-Methionine Labeling of Yeast Cells

35S-Methionine Labeling of Yeast Cells
Contributor: The Laboratory of Andrew Murray at Harvard University
 
Overview
This is a general protocol for the metabolic labeling of proteins with 35S in actively growing yeast cells.
 
Procedure
1. Grow a 20 ml culture of yeast cells into the mid-logarithmic growth range to an Optical Density at 600 nm (OD600) of 0.3 and 0.6 (see Hint #1).

2. Harvest the cells by transferring the culture to a 50 ml polypropylene conical tube and centrifuging for 5 min at 1,500 X g. Discard the supernatant.

3. Resuspend the pellet of cells in 10 ml of Minimal Medium Lacking Methionine.

4. Pellet the cells again by centrifugation for 5 min at 1,500 X g. Discard the supernatant.

5. Resuspend the cells in 3 ml of Minimal Medium Lacking Methionine.

6. Add 25 μCi of 35S-Methionine per OD600 units of cells (CAUTION! see Hint #2).

7. Incubate cells with 35S-Methionine for 30 min to 1 hr with gentle rotary shaking.

8. Pellet the cells by centrifuging for 5 min at 1,500 X g. Discard the supernatant in the liquid radioactive waste receptacle.

9. The cells are now ready for analysis of labeled proteins, or the cells can be frozen in Liquid Nitrogen and stored at -80°C until future analysis.

Solutions
35S-Methionine   1,000 Ci/mmol
Minimal Medium Lacking Methionine   20 g Glucose
6.7 g Yeast Nitrogen Base (without Amino Acids)
Autoclave
Make up to 1 liter with ddH2O
Add necessary Amino Acid Supplements
 
BioReagents and Chemicals
35S-Methionine
Amino Acid Supplements
Yeast Nitrogen Base, Amino Acid Free
Glucose
Yeast
Nitrogen, Liquid
 
Protocol Hints
1. Depending on the growth media used, the growth rate of the cells and the optical density of mid-log growth may vary.

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