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MOLECULAR BIOLOGY: WORKING WITH DNA

PROTEIN EXPRESSION: LABELING

Overexpression of Recombinant Proteins in COS-7 Cells

Overexpression of Recombinant Proteins in COS-7 Cells
 
Overview
This protocol describes how to perform a DEAE-dextran transfection of COS cells and provides information on labeling the expressed protein with [35S-Cysteine] for SDS-PAGE analysis.
 
Procedure
A. Transfection of COS-7 Cells with DEAE-Dextran

1. Prepare a 1:30 dilution of the DEAE-dextran in 1X TBS.

2. Dilute the plasmid constructs in 1X TBS. Use 15 μg of plasmid DNA per 10 cm tissue culture dish. Dilute 15 μg of DNA into 750 μl TBS (see Hint #1).

3. Add one volume of the diluted DEAE-dextran from Step #A1 to three volumes of the DNA solution from Step #A2.

4. Take COS-7 cells grown in DMEM Complete Medium to approximately 60% confluency (600,000 cells/plate) and rinse with TBS.

5. Evenly cover the plate with 1 ml of the DNA-Dextran mixture from Step #A3.

6. Incubate for 45 min at room temperature, gently rocking every 10 min to ensure that no area on the plate dries out.

7. Aspirate the DNA-dextran mixture and add 6 ml of Chloroquine Solution per plate.

8. Incubate at 37°C for 4 hr or until the cells are distinctly studded with vacuoles and are beginning to detach from the plates.

9. Replace the Chloroquine Solution with 10 ml DMEM Complete Medium and incubate at 37°C overnight.

10. Replace the DMEM Complete Medium with Serum-free Medium and incubate at 37°C for 3 days.

11. Harvest the media (this media is called the conditioned media).

12 Concentrate the conditioned media though a 10 kDa cut-off centrifugal filter (Centriprep-10, Amicon) to approximately 500 μl for subsequent assays.

B. Metabolic Labeling of Proteins with [35S]-Cysteine

1. Perform Steps #1-#9 of Section A with a 6-well flat-bottom tissue culture plate and scale down all volumes five-fold.

2. Two days after the transfection, measure the transfection efficiency by performing a Beta-Galactosidase assay (see Protocol ID#209 and see Hint #1).

3. Rinse each well with Serum-Free Medium and add 1 ml of Labeling Cocktail to each well.

4. Incubate overnight at 37°C.

5. Harvest and concentrate the media as in Steps #A11 and #A12.

6. Add an equal volume of 2X Protein Sample Buffer.

7. Load the samples onto a 13% denaturing SDS gel and run the gel (see Protocol ID#455).

8 Fix the gel in Acetic Acid/Methanol for 30 min at room temperature.

9. Incubate gel in Amplify (Amersham) for 15 min to enhance the [35S] signal.

10. Dry the gel under vacuum.

11. Expose to film at -70°C (for 2 hr to overnight) and develop for evaluation of expression levels by densitometry.

Solutions
Protein Sample Buffer (2X)   Add DTT just before use.
2 mM DTT
20% (v/v) Glycerol
100 mM Tris-Cl, pH 6.8
0.2% (w/v) Bromophenol Blue
4% (w/v) SDS
Acetic Acid/Methanol   30% (v/v) Acetic Acid
10% (v/v) Methanol
Labeling Cocktail (100X)   100 mCi/ml [35S]-Cysteine (Amersham; CAUTION! See Hint #2)
Prepare in Minimal Essential Medium (MEM) lacking Cysteine
Serum-Free Medium   DMEM
10 mg/ml Transferrin
5 mg/ml Insulin
DEAE-dextran   100 mg/ml DEAE-dextran
Filter sterilize
Chloroquine Solution   Prepare in DMEM Complete Medium
100 mM Chloroquine
DMEM Complete Medium   2 mM Glutamine
Dulbecco's Modified Eagle's Medium (DMEM)
60 mg/ml Gentamycin
10% (v/v) Fetal Calf Serum
TBS   Keep sterile
890 ml sterile ddH2O
Add 10 ml of Solution B drop-wise while stirring
100 ml Solution A
Solution B   27 mM CaCl2
21 mM MgCl2
Autoclave
Solution A   Autoclave
1.37 M NaCl
14 mM Sodium Phosphate Dibasic (Na2HPO4)
50 mM KCl
250 mM Tris
 
BioReagents and Chemicals
Glutamine
Potassium Chloride
Sodium Chloride
Acetic Acid
DTT
Bromophenol Blue
SDS
Methanol
Fetal Calf Serum
[35S]-Cysteine
Calcium Chloride
DMEM
Transferrin
DEAE-dextran
Cloroquine
Amplify
Sodium Phosphate, Dibasic
Glycerol
Magnesium Chloride
Gentamycin
Insulin
Tris
MEM
 
Protocol Hints
1.Use a control plasmid (15 μg) like pCH110 that expresses Β-galactosidase to evaluate transfection efficiency. If the assay shows an absorbance reading at 420 nm of 0.5 and above, the transfection is considered good.

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