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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: TRANSFECTION

Overexpression of Recombinant Proteins in COS-7 Cells

Overexpression of Recombinant Proteins in COS-7 Cells Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

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 Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

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 Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

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 Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

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 Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

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.