Western Blotting and Molecular Biology: Western Blot Protocol (modified)

A. Preparation of cell lysates

Harvest cells (70-85% confluent) by trypsinization and spin.
Lyse the pellet with 100 µl lysis buffer on ice for 10 min.

Note: 1) Just scrub the cells after washing with PBS and adding the lysis buffer, if the cells being plated at dishes or plates.

2) Sonication is optional after lysing the sample, but just don’t sonicate the sample too long. We suggest it should be sonicated for no more than 10 seconds.

3) The amount of lysis buffer is flexible. It depends on the number of cells you have.

4) You need to homogenize it first if you do Western blotting with tissue.

Spin at 14,000 rpm (16,000 g) in an Eppendorf microfuge for 20 min at 4°C.
Transfer the supernatant to a new tube and discard the pellet.
Measure the protein concentration with protein assay whichever method set up by your lab. (e.g. Bradford assay, or BCA; or Lowry Assay)

Note: Don’t dilute the protein too much. You can concentrate the protein lysate with vacuum under 4ºC in case you got a very low protein concentration.

Take the same amount protein lysate from each sample separately, and mix with sample loading buffer. The final concentration of loading buffer must be 1X.

e.g. The ratio of sample (ul)/ 2X loading buffer (ul) should be 1:1. The ratio of sample (ul)/ 6X loading buffer (ul) should be 5:1.

Boil for 5 min.
Cool at room temperature (RT) for 5 min.
Briefly spin to bring down the sample and loading buffer mixture prior to loading gel.

B. Polyacrylamide gel (14.5 cm x 16.5 cm)

Agarose plug:
1% agarose dissolved in 1x Resolving gel buffer.
(I make 50 ml, keep melting it as you need it, and re-adding water to maintain agarose conc.)
Resolving gel: 24 ml of a 9% gel
5.4 ml 40% acrylamide/bisacrylamide (29:1 mix)
3 ml 8x Resolving gel buffer
15.6 ml water
12 µl TEMED
60 µl 20% ammonium persulfate
Stacking gel: 8 ml
1 ml 40% acrylamide/bisacrylamide (29:1 mix)
2 ml 4x Stacking gel buffer
5 ml water
8 µl TEMED
21.6 µl 20% ammonium persulfate

C. Preparation of gel

Assemble the glass plates and spacers (1.5 mm thick).
Pour an agarose plug (1-2 mm).
Pour the running gel to about 1 cm below the wells of the comb (~20 ml).
Seal with 1 ml water-saturated 1-butanol.
(Can stop here and leave gel as is overnight if you want.)
When gel has set, pour off the butanol and rinse with deionized water.
Pour the stacking gel (~5 ml) and insert the comb immediately.
When the stacking gel has set, place in gel rig and immerse in buffer.
Prior to running the gel, flush the wells out thoroughly with running buffer.

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