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

MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN DETECTION: GEL ELECTROPHORESIS

SDS-PAGE SAMPLE PREPARATION OF ADENOVIRUS INFECTED CELLS

SDS-PAGE Sample Preparation of Adenovirus Infected Cells
Contributor: The Laboratory of Daniel Portnoy at the University of California, Berkeley
 
Procedure
1. Infect a confluent 100 mm plate of HEK 293 cells with the desired adenovirus.

2. Allow the infection to completely lyse the monolayer of cells.

3. After harvesting cells and media, place them into a 15 ml conical tube.

4. Centrifuge the cells for 4 min at 1,000 X g in a clinical centrifuge.

5. Aspirate the supernatant.

6. Remove the supernatant and resuspend the cell pellet in 10 ml of PBS.

7. Centrifuge the cells for 4 min at 1,000 X g in a clinical centrifuge.

8. Aspirate the supernatant.

9. Wash the cells again in 10 ml of PBS and centrifuge the cells for 4 min at 1,000 X g in a clinical centrifuge.

10. Aspirate the supernatant and resuspend the cell pellet in 100 μl of PBS.

11. Transfer the cell suspension to a microcentrifuge tube.

12. Add 100 μl of 2X SDS Laemli Buffer and mix thoroughly with pipette in up-and-down strokes.

13. Sonicate the cell suspension with short bursts of a sonic cell disrupter until the cells are lysed (see Hint #1).

14. Incubate in a boiling-water bath for 3 minutes.

15. Run the sample on a SDS polyacrylamide gel (see Protocol ID#455).

Solutions
SDS Laemli Buffer (2X)   20% (v/v) Glycerol
100 mM Tris-HCl, pH 6.8
10% (v/v) 2-mercaptoethanol
4% (w/v) SDS
0.2% (w/v) Bromophenol Blue
PBS   pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
 
BioReagents and Chemicals
Sodium Phosphate, Dibasic
Tris
HEK 293 Cells
2-Mercaptoethanol
Potassium Chloride
Sodium Chloride
Bromophenol Blue
Glycerol
SDS
Potassium Phosphate, Monobasic
 
Protocol Hints
1. Check for complete cell lysis under a microscope.