Modified Protocol for Western Blotting (Page 1, 2, 3)--Western Blotting Center
D. Running the gel
- After briefly spinning
the samples, load into the wells.
- Be sure to use protein markers.
- Run with constant current or voltage. (We usually run the gel with
100v constant voltage first, and then adjust according to the shift of bands.)
- Usual running time is about 1.5-2.5 hrs.
E. Using precast
gels (Ready Gels):
- Assemble gel in gel rig.
- Prepare protein samples.
- Use a standard protein marker.
- Run with power supply at 100v
first and adjust according to the shift of bands.
F. Preparation of membrane
- Cut a piece of polyvinylidene difluoride (PVDF) membrane.
- Wet for about 30 min in methanol on a rocker at room temp.
- Remove methanol and add 1x Blotting buffer until ready to use.
Note: Just
wet it with cooled blotting buffer for 30 minutes, if you select nitrocellulose.
G. Membrane transfer
- Assemble "sandwich" using a transfer.
- Prewet the sponges, filter papers (slightly bigger than gel) in 1x Blotting buffer.
Sponge - filter
paper - gel - membrane - filter paper - sponge
- Transfer for 1 hr at 1 amp constant current (or 100 voltage constant voltage) at 4°C
on a stir plate.
Bigger proteins might take longer to transfer.
Note: the time or current (or voltage)
is different from lab to lab. You can change them according to the apparatus you are using and your experience at the lab.
- When finished,
immerse membrane in Blocking buffer and block at 4ºC overnight, or one hour at room temperature.
Note: In order to get a good blot,
we suggest to make the transferring buffer freshly every time you do Western blotting.
H. Antibodies and detection
- Incubate with
primary antibody diluted in Blocking buffer (usually, 5% milk) for 60 min at room temp, or overnight at .4°C.
- Wash 3 x 5 min with
0.1% Tween 20 in PBS (TBST).
- Incubate with secondary antibody diluted in Blocking buffer for one hour at room temp.
- Wash 3 x 10 min
( or 4 x 5 min) with TBST.
- Detect with Enhanced Chemiluminescence (ECL).
Note: If you want to look into both non-phospho and phosphor-protein
with the same blot, make sure to probe the phosphor-antigen first, and be sure you add proper phosphotase inhibitor during the Western
blotting procedure due to the high activity of phosphotase. I usually use phosphotase inhibitor cocktail and 50mM sodium floride.
I.
Stripping blot
- Rinse blot off with TBST.
- Put blot into Kapak bag cut to slightly bigger size than blot.
- Add about 5 to 10 ml Stripping
buffer.
- Remove as much air as possible and seal bag.
- Incubate for 20 min with shaking at 37ºC.
- Rinse blot off with TBST.
- Repeat step
H (1-5) as above.