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MOLECULAR BIOLOGY: WORKING WITH DNA

PROTEIN EXPRESSION: LABELING

Steady State Labeling with [35S] Trans Label in Drosophila L2 Cells

Steady State Labeling with [35S] Trans Label in Drosophila L2 Cells
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
 
Procedure
1. Centrifuge 200 ml of L2 cells (from a confluent culture; 2.5 million cells/ml) in 50 ml tubes in a tabletop centrifuge at a setting of 5 (1,000 X g) for 3 min (see Hint #2)

2. Remove the media in a sterile hood and resuspend the cells in 40 ml (10 ml/tube) of PBS with a wide-bore 10 ml pipette and transfer all the cells into a single 50 ml tube.

3. Centrifuge in a tabletop centrifuge at a setting of 5 (1,000 X g) for 3 min and remove the PBS.

4. Resuspend the cells in 1.5 ml of M3 Insect Medium and transfer to a small (30 mm) petri dish.

5. Add 1 mCi of [35S] Trans label and incubate overnight at 25°C. (CAUTION! see Hint #1)

7. After the overnight labeling, transfer the cells from the plate to a 15 ml tube. Wash off the plate with 10 ml of PBS and add that to the 15 ml tube as well.

8. Centrifuge the tube in a clinical tabletop centrifuge at a setting of 5 (1,000 X g) for 3 min. Determine the packed cell volume (PCV) (see Hint #3). It should be approximately 0.5 ml.

9. Remove the supernatant and resuspend the cells in 15 ml of PBS, using a Pasteur pipette to aid in the resuspension if necessary.

10. Centrifuge the tube in a clinical tabletop centrifuge at a setting of 5 (1,000 X g) for 3 min. Determine the packed cell volume (PCV). It should now be approximately 0.3 ml.

11. Resuspend the cells in 3 times the PCV (probably about 0.9 ml) with Cell Lysis Buffer and transfer the suspension to a 1.5 ml microcentrifuge tube.

12. Incubate the solution on ice for 5 min.

13. Centrifuge at full speed in a microcentrifuge for 10 min at 4°C.

14. Recover the supernatant and transfer this [35S]-labeled cell extract to a fresh tube. Keep the extract on ice until freezing it in Liquid Nitrogen. Discard the pellet, which is cell debris.

15. Mix 1 μl of the cell extract and 5 ml of Scintillation Fluid well in a small scintillation vial and count in an [35S] channel in a scintillation counter. Expect counts greater than 200,000 cpm/μl of extract. One million cpm per μl extract is excellent.

16. Store the extract at -80°C.

Solutions
70 mM Copper Sulfate (CuSO4)
PBS   pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
137 mM NaCl
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
Cell Lysis Buffer   200 mM LiCl
0.1 mg/ml Phenylmethylsulfonyl Fluoride (PMSF; CAUTION! see Hint #1)
50 mM Tris-HCl, pH 8.0
0.5% (v/v) Igepal CA-630 (replaces NP-40)
10 mM Sodium Metabisulfite (Na2S2O5)
1 mM EDTA
Add PMSF and Na2S2O5 to buffer right before use.
 
BioReagents and Chemicals
Scintillation Fluid
[35S] Trans-Label
Tris
Potassium Phosphate, Monobasic
EDTA
Sodium Phosphate, Dibasic
Lithium Chloride
Potassium Chloride
Sodium Metabisulfite
Sodium Chloride
M3 Insect Medium
PMSF
IGEPAL CA-630
Nitrogen, Liquid
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. The original protocol utilized a cell line containing a gene of interest under the control of the metallothionine promoter.

3. The Packed Cell Volume can be determined by filling an empty tube with water to the same volume as the cell pellet occupies, as judged by eye. Measure the volume of water. That is the PCV.