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Western Blot Protocol

Buffers:

RIPA buffer:

Phosphate-buffered saline (PBS)

Adjust pH to 7.2-7.4. Autoclave before use.

293T Cell lysate preparation:

1. Carefully aspirate culture media from each well of the 6-well plate.
2. Add 1mL PBS to wash each well and remove the wash.
3. Add 1mL PBS to each well and resuspend the cell by pipetting up and down multiple times.
4. Transfer the 1mL resuspension to a clean EP tube and label well.
5. Centrifuge at 4oC at 800g for 10min to spin down the cells and remove the supernatant.
6. Add 400ul RIPA buffer to each tube and pipette several times to break the pellet.
7. Rock the tube at 4oC overnight to lysis the cell.

SDS-PAGE:

1. Proper amount of cell lysate was mixed with 5X loading buffer and boiled for 5 minutes before loading.
2. Run the gel at 50V for 5 minutes.
3. Invrease the voltage to 100-150V and run for 1-2 hours.

Transfer the protein to PVDF membrane:

1. The PVDF membrane was soaked in methonal immediately before use.
2. Assemble the transfer sandwich with clip, fiber pad, filter paper, gel and membrane.
3. Put the transfer sandwich into transfer buffer and transfer on ice at 100v for 1h.

Blocking:

1. Block the membrane with 5% non-fat milk for at room temperature for at least 2h.

Antibody incubation:

1. Incubate the membrane with primary antibody at proper dilution in TTBS for 2h or at 4oC overnight.
2. Wash the membrane with TTBS: 3x10 minutes.
3. Incubate the membrane with secondary antibody conjugated with horse radsih peroxidase at proper dilution in TTBS for 2h.
4. Wash the membrane with TTBS: 3x10 minutes.

Horse radish peroxidase(HRP) staining and imaging:

1. Incubate the membrane with HRP substrate following manufacturer's instructions.
2. Expose to film and develop image.

 

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