Please find the following protocol:

SDS-PAGE Sample Preparation of Adenovirus Infected Cells



Protocol:

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. Check for complete cell lysis under a microscope.

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

15. Run the sample on a SDS polyacrylamide gel



Solutions:

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

SDS Laemli Buffer (2X)...............................20% (v/v) Glycerol
........................100 mM Tris-HCl, pH 6.8
................10% (v/v) 2-mercaptoethanol
................0.2% (w/v) Bromophenol Blue
......................................4% (w/v) SDS



Bioreagents and Chemicals:

Glycerol
SDS
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
Tris
HEK 293 Cells
2-Mercaptoethanol
Potassium Chloride
Sodium Chloride
Bromophenol Blue


Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p1752